Klk5 inhibitory peptide

ABSTRACT

SPINK2 mutant peptide conjugates are provided that inhibit KLK5. The KLK5 inhibitory peptide conjugates are Fc fusion peptides in which, in certain embodiments, the Fc region of the fusion peptides are the Fc region of human IgG1 or a fragment thereof. The KLK5 inhibitory peptide conjugates include an amino acid sequence of one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, 96, 50, 52, 54, 56, 58, or 60. Pharmaceutical compositions that include the KLK5 inhibitory peptide conjugates useful for treating KLK5-related diseases are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 17/064,543,filed Oct. 6, 2020, which is a continuation of International ApplicationNo. PCT/JP2019/043384, filed Nov. 6, 2019, which claims priority toJapanese Application No. 2018-209729, filed Nov. 7, 2018, each expresslyincorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is DAISAN174089_Seq_List_FINAL_20210519_ST25.txt.The text file is 155 KB; was created on May 19, 2021; and is beingsubmitted via EFS-Web with the filing of the specification.

TECHNICAL FIELD

The present invention relates to a peptide, a polynucleotide, a vector,a cell, a method for producing the peptide, a peptide obtained by such amethod, a conjugate containing the peptide, a composition containing thepeptide or the conjugate, a pharmaceutical composition, thepharmaceutical composition for treating or preventing various diseases,use of the peptide or the conjugate for treating or preventing variousdiseases, a method for treating various diseases that includes the stepof administering the peptide or the conjugate, a composition fordiagnosing or testing various diseases that contains the peptide or theconjugate, and the like.

BACKGROUND ART

Kallikrein 5 (KLK5) is a trypsin-like serine protease (Clan PA, familyS1) and is also called a stratum corneum tryptic enzyme (SCTE).Kallikrein 7 (KLK7) is a chymotrypsin-like protease (Clan PA, family S1)and is also called a stratum corneum chymotryptic enzyme (SCCE).Further, kallikrein 14 (KLK14) is a trypsin-like protease (Clan PA,family S1). KLK5, KLK7, and KLK14 belong to a tissue kallikrein familycomposed of 15 types of highly conserved trypsin- or chymotrypsin-likeserine proteases. KLK5 is expressed in cells and thereafter convertedinto active KLK5 by autoactivation, whereas KLK7 and KLK14 are expressedas inactive preproenzymes and converted into active forms by cleavage oftheir preprosequences by proteases represented by KLK5 (Non PatentLiterature 1). Expression of KLK5 has been observed in the skin, and itis reported to degrade factors related to cell adhesion such asDesmoglein and Desmocollin (Non Patent Literature 2). KLK5, KLK7, andKLK14 are important for skin desquamation and are further involved inactivation of proteinase-activated receptor 2 (PAR-2) (Non PatentLiterature 3). Activation of PAR-2 induces cytokines and chemokines andenhances immunity, inflammation reactions, and the like.

Netherton syndrome is one of the ichthyosis syndromes with severe skininflammation, desquamation, abnormal hair, and allergic symptoms such asasthma and allergic dermatitis due to autosomal recessive inheritance,and is a rare disease (0=256500) (Non Patent Literature 4). Sinceexfoliative dermatitis develops from the time of birth, dehydration,infection, or the like may be caused by significant damage to the skinbarrier function and may be accompanied by developmental delay. Althoughdetailed epidemiological data is not available, it is said that highpostnatal mortality is exhibited. Netherton syndrome develops due to theloss of function of a serine protease inhibitor (LEKTI) following amutation of a gene (SPINK5) encoding LEKTI expressed in skin epithelialcells. LEKTI is composed of 15 Kazal-like inhibitor domains. In patientswith Netherton syndrome, a plurality of mutation sites has been found inSPINK5 across the sequence encoding each domain, and symptoms andseverity change in association with the mutation sites (Non PatentLiteratures 4 and 5).

SPINK5-deficient mice (Spink5^(−/−)) exhibit Netherton syndrome-likeskin symptoms, and high protease activity of KLK5 and KLK7 is observedin the skin epithelium (Non Patent Literature 1). AlthoughSPINK5-deficient mice die within several hours after birth, improvementof neonatal mortality is reported in mice (Spink5^(−/−)Klk5^(−/−))obtained by crossing KLK5-deficient mice with SPINK5-deficient mice (NonPatent Literature 6). Further, in Spink5^(−/−)Klk5^(−/−)mice, severeskin barrier defects, epithelium structural defects, skin inflammation,and the like observed in Spink5^(−/−)have been recovered. Likewise, micewith the SPINK5 mutation Spink5^(A135X/A135X) observed in patients withNetherton syndrome show Netherton syndrome-like skin symptoms, and deathis observed within 12 hours after birth, whereas skin barrier and severeskin symptoms are improved in Klk5^(−/−)Spink5^(A135X/A135X) obtained bycrossing with KLK5-deficient mice (Non Patent Literature 7). Further,abnormal skin symptoms are eliminated by crossing with KLK7-deficientmice (Klk5^(−/−)Klk7^(−/−)Spink5^(A135X/A135X)). It has been reportedthat KLK5 transgenic mice also show Netherton syndrome-like skinsymptoms (Non Patent Literature 8). High protease activity like trypsinand chymotrypsin is observed in the stratum corneum of patients withNetherton syndrome and Netherton syndrome model mice, and it issuggested that the kallikrein family located downstream, such as KLK7and KLK14, are related to the protease activity in the stratum corneum,in addition to KLK5. From the above, it is considered that Nethertonsyndrome is caused by a gene mutation of SPINKS and develops withabnormally elevated KLK5, KLK7, or KLK14 protease activity in thecorneum. Currently, there is no fundamental therapeutic agent but onlysymptomatic treatment such as application of humectants.

KLK5 has also been suggested to be associated with rosacea, which is achronic inflammatory disease of the face. In rosacea patients, increasedexpressions of KLK5 and the antimicrobial peptide Cathelicidin have beenreported. Although the details of its etiology are unknown, it isconsidered that KLK5 with elevated expression degrades Cathelicidin,thereby producing a peptide that causes rosacea (Non Patent Literature9).

There are multiple reports that SPINK5 polymorphisms are associated withthe severity of atopic dermatitis (Non Patent Literatures 10 to 14). Ithas been reported that, in the skin of atopic dermatitis patients havingSPINK5 genes encoding LEKTI in which amino acid residue 420 is lysine inboth alleles, the expression of Desmogleinl is decreased at the proteinlevel, and the activity of proteases, including KLK5 and KLK7, isincreased, as compared with the case in which glutamic acid is encodedin both alleles (Non Patent Literature 15). It is considered that theactivation of these proteases facilitates the invasion of allergens dueto a decrease in skin barrier function and creates a condition where aninflammation reaction easily occurs.

SPINK2 (Serine Protease Inhibitor Kazal-type 2) is composed of aKazal-like domain having three disulfide bonds and functions as atrypsin/acrosin inhibitor (Non Patent Literature 16), but therelationship of SPINK2 and its mutants to diseases such as Nethertonsyndrome, rosacea, and atopic dermatitis has not been clarified.

CITATION LIST Non Patent Literatures

-   Non Patent Literature 1: Ovaere P, et al., published in 2009, Trends    Biochem Sci., Vol. 34, No. 9: pp. 453-463-   Non Patent Literature 2: Descargues P, et al., published in 2005,    Nat Genet., Vol. 37, No. 1: pp. 56-65-   Non Patent Literature 3: Rattenholl A, et al., published in 2008,    Drug News Perspect., Vol. 21, No. 7: pp. 369-381-   Non Patent Literature 4: Hovnanian A., published in 2013, Cell    Tissue Res., Vol. 351, No. 2: pp. 289-300-   Non Patent Literature 5: Sarri C A, et al., published in 2017, Mol    Diagn Ther., Vol. 21, No. 2: pp. 137-152-   Non Patent Literature 6: Furio L, et al., published in 2015, PLoS    Genet., Vol. 11, No. 9: e1005389-   Non Patent Literature 7: Kasparek P, et al., published in 2017, PLoS    Genet., Vol. 13, No. 1: e1006566-   Non Patent Literature 8: Furio L, et al., published in 2014, J Exp    Med., Vol. 211, No. 3: pp. 499-513-   Non Patent Literature 9: Yamasaki K, et al., published in 2007, Nat    Med., Vol. 13, No. 8: pp. 975-980-   Non Patent Literature 10: Nishio Y, et al., published in 2003, Genes    Immun., Vol. 4, No. 7: pp. 515-517-   Non Patent Literature 11: Kusunoki T, et al., published in 2005, J    Allergy Clin Immunol., Vol. 4, No. 7: pp. 515-517-   Non Patent Literature 12: Lan C C, et al., published in 2011, Exp    Dermatol., Vol. 20, No. 12: pp. 975-979-   Non Patent Literature 13: Kato A, et al., published in 2003, Br J    Dermatol., Vol. 148, No. 4: pp. 665-669-   Non Patent Literature 14: Zhao L P, et al., published in 2012, J Eur    Acad Dermatol Venereol., Vol. 26, No. 5: pp. 572-577-   Non Patent Literature 15: Fortugno P, et al., published in 2012, Hum    Mol Genet., Vol. 21, No. 19: pp. 4187-4200-   Non Patent Literature 16: Chen T, et al., published in 2009,    Proteins., Vol. 77, No. 1: pp. 209-219

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a novel KLK5 inhibitorypeptide, a conjugate containing the peptide, a pharmaceuticalcomposition containing the peptide or the conjugate, and the like.

Solution to Problem

The present invention relates to:

(1) A SPINK2 mutant peptide that comprises the amino acid sequence setforth in SEQ ID NO: 61 (FIG. 69) and inhibits the protease activity ofactive human KLK5;(2) The peptide according to (1), wherein the peptide inhibits theprotease activity of human KLK7 or human KLK14;(3) The peptide according to (1), wherein the peptide selectivelyinhibits human KLK5 and optionally human KLK7 or KLK14;(4) The peptide according to any one of (1) to (3), wherein Xaa16 (X₁)is Ala, Asp, Gly, Gln, Leu, Ser, or Thr;(5) The peptide according to any one of (1) to (4), wherein Xaa17 (X₂)is Arg, Glu, Asn, Gln, or Ser;(6) The peptide according to any one of (1) to (5), wherein Xaa18 (X₃)is Asp, Gln, Ile, Thr, Trp, or Tyr;(7) The peptide according to any one of (1) to (6), wherein Xaa19 (X₄)is Arg, Gly, Met, Gln, or Thr;(8) The peptide according to any one of (1) to (7), wherein Xaa20 (X₅)is Asp, Glu, Leu, Lys, Thr, or Tyr;(9) The peptide according to any one of (1) to (8), wherein Xaa21 (X₆)is Glu, Gly, His, Leu, Ser, Gln, or Tyr;(10) The peptide according to any one of (1) to (9), wherein Xaa22 (X₇)is Asp, Gly, Gln, Ser, or Tyr;(11) The peptide according to any one of (1) to (10), wherein Xaa24 (X₈)is Ala, Asp, Glu, Gly, Asn, Ser, or Thr;(12) The peptide according to any one of (1) to (11), wherein Xaa25 (X₉)is Arg or Lys;(13) The peptide according to any one of (1) to (12), wherein Xaa26(X₁₀) is Asp, Glu, Gln, Ser, or Val;(14) The peptide according to any one of (1) to (13), wherein Xaa27(X₁₁) is Phe or Tyr;(15) The peptide according to any one of (1) to (14), wherein Xaa28(X₁₂) is Asp or Glu;(16) The peptide according to any one of (1) to (15), wherein thepeptide comprises the amino acids at positions 1 to 63 in the amino acidsequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18,and 20 (FIGS. 14, 16, 18, 20, 22, 24, 26, and 28);(17) The peptide according to any one of (1) to (3), wherein Xaa16 (X₁)is Gly, Met, or Tyr;(18) The peptide according to any one of (1) to (3) and(17), wherein Xaa17 (X₂) is Glu, Gln, or Thr;(19) The peptide according to any one of (1) to (3), (17), and (18),wherein Xaa18 (X₃) is His, Met, or Tyr;(20) The peptide according to any one of (1) to (3) and (17) to (19),wherein Xaa19 (X₄) is Ala, Arg, Lys, or Gln;(21) The peptide according to any one of (1) to (3) and (17) to (20),wherein Xaa20 (X₅) is Gly, Arg, or Ser;(22) The peptide according to any one of (1) to (3) and (17) to (21),wherein Xaa21 (X₆) is Arg, Lys, Gln, or Ser;(23) The peptide according to any one of (1) to (3) and (17) to (22),wherein Xaa22 (X₇) is Gly;(24) The peptide according to any one of (1) to (3) and (17) to (23),wherein Xaa24 (X₈) is His, Thr, or Tyr;(25) The peptide according to any one of (1) to (3) and (17) to (24),wherein Xaa25 (X₉) is His or Tyr;(26) The peptide according to any one of (1) to (3) and (17) to (25),wherein Xaa26 (X₁₀) is Asp, Glu, or His;(27) The peptide according to any one of (1) to (3) and (17) to (26),wherein Xaa27 (X₁₁) is Tyr;(28) The peptide according to any one of (1) to (3) and (17) to (27),wherein Xaa28 (X₁₂) is Asp or Glu;(29) The peptide according to any one of (1) to (3) and (17) to (28),wherein the peptide comprises the amino acids at positions 1 to 63 inthe amino acid sequence set forth in any one of SEQ ID NOs: 22, 24, 26,and 28 (FIGS. 30, 32, 34, and 36);(30) The peptide according to any one of (1) to (3), wherein Xaa16 (X₁)is Gly, Ser, or Tyr;(31) The peptide according to any one of (1) to (3) and (30), whereinXaa17 (X₂) is Asp or Gln;(32) The peptide according to any one of (1) to (3), (30), and (31),wherein Xaa18 (X₃) is Thr or Val;(33) The peptide according to any one of (1) to (3) and (30) to (32),wherein Xaa19 (X₄) is Thr or Val;(34) The peptide according to any one of (1) to (3) and (30) to (33),wherein Xaa20 (X₅) is Glu or Thr;(35) The peptide according to any one of (1) to (3) and (30) to (34),wherein Xaa21 (X₆) is His or Thr;(36) The peptide according to any one of (1) to (3) and (30) to (35),wherein Xaa22 (X₇) is Tyr;(37) The peptide according to any one of (1) to (3) and (30) to (36),wherein Xaa24 (X₈) is Asn or Ser;(38) The peptide according to any one of (1) to (3) and (30) to (37),wherein Xaa25 (X₉) is Arg;(39) The peptide according to any one of (1) to (3) and (30) to (38),wherein Xaa26 (X₁₀) is Asp or Glu;(40) The peptide according to any one of (1) to (3) and (30) to (39),wherein Xaa27 (X₁₁) is Tyr;(41) The peptide according to any one of (1) to (3) and (30) to (40),wherein Xaa28 (X₁₈) is Asp;(42) The peptide according to any one of (1) to (3) and (30) to (41),wherein the peptide comprises the amino acids at positions 1 to 63 inthe amino acid sequence set forth in any one of SEQ ID NOs: 30 and 32(FIGS. 38 and 40);(43) The peptide according to any one of (1) to (42), wherein thepeptide has three disulfide bonds and has a three-dimensional structurecharacterized by including a loop structure, an α-helix, and a β-sheet;(44) A polynucleotide comprising a nucleotide sequence encoding theamino acid sequence of the peptide according to any one of (1) to (43);(45) A vector comprising the polynucleotide according to (44);(46) A cell that comprises the polynucleotide according to (44) or thevector according to (45) or produces the peptide according to any one of(1) to (43);(47) A method for producing a SPINK2 mutant peptide, comprising thesteps (i) and (ii) below: (i) culturing the cell according to (46); and(ii) collecting the SPINK2 mutant peptide from the culture;(48) A method for producing the peptide according to any one of (1) to(43), comprising a step of preparing the peptide by chemical synthesisor in-vitro translation;(49) A SPINK2 mutant peptide obtained by the method according to (47) or(48);(50) A conjugate comprising the peptide according to any one of (1) to(43) and (49) wherein the peptide is bound by one and optionally morethan one moiety;(51) The conjugate according to (50), wherein the one or more optionalmoiety comprises a second peptide that is not the SPINK2 mutant;(52) The conjugate according to (51), wherein the second peptide islocated on the amino terminal side of the SPINK2 mutant;(53) The conjugate according to (51), wherein the second peptide islocated on the carboxyl terminal side of the SPINK2 mutant;(54) The conjugate according to any one of (51) to (53), wherein thesecond peptide is an antibody or a fragment thereof and comprises one ormore Fc regions;(55) The conjugate according to (54), wherein the or each Fc region isan Fc region of human immunoglobulin, or a fragment thereof;(56) The conjugate according to (54) or (55), wherein the or each Fcregion is an Fc region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD,and/or IgE, or a fragment thereof;(57) The conjugate according to any one of (54) to (56), wherein the oreach Fc region is an Fc region of human IgG1, or a fragment thereof;(58) The conjugate according to (57), wherein the or each Fc region ofhuman IgG1 comprises the amino acid sequence set forth in SEQ ID NO: 87(FIG. 95);(59) The conjugate according to any one of (54) to (57), wherein the oreach Fc region is a wild type or a mutant Fc region;(60) The conjugate according to any one of (51) to (59), wherein theconjugate comprises one to several aspartic acids and/or glutamic acidsadded to the amino terminus thereof;(61) The conjugate according to any one of (50) to (60), wherein theconjugate comprises the amino acid sequence described in (i) or (ii)below: (i) the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 96 (FIGS.42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 106); or(ii) the amino acid sequence of a conjugate that is 90% or moreidentical to the amino acid sequence described in (i) above and inhibitsthe protease activity of KLK5;(62) The conjugate according to any one of (51) to (61), wherein theSPINK2 mutant and the second peptide are linked to each other via alinker;(63) The conjugate according to (62), wherein the linker is a thirdpeptide that is not the SPINK2 mutant or the second peptide;(64) A method for producing the conjugate according to any one of (50)to (63), comprising the steps (i) and (ii) below: (i) culturing a cellcomprising a polynucleotide comprising a nucleotide sequence encoding anamino acid sequence of the conjugate or a vector in which thepolynucleotide is inserted; and (ii) collecting a SPINK2 mutant peptideconjugate or a peptide moiety comprised in the conjugate from theculture;(65) A method for producing the SPINK2 mutant peptide conjugateaccording to any one of (50) to (63), comprising a step of preparing theconjugate or a peptide moiety comprised in the conjugate by chemicalsynthesis or in-vitro translation;(66) A SPINK2 mutant peptide conjugate obtained by the method accordingto (64) or (65);(67) An antibody or a binding fragment thereof that binds to the peptideaccording to any one of (1) to (43) and (49);(68) A composition comprising the peptide according to any one of (1) to(43) and (49), the polynucleotide according to (44), the vectoraccording to (45), the cell according to (46), the conjugate accordingto any one of (50) to (63) and (66), and/or the antibody or the bindingfragment thereof according to (67);(69) A pharmaceutical composition comprising the peptide according toany one of (1) to (43) and (49), the polynucleotide according to (44),the vector according to (45), the cell according to (46), and/or theconjugate according to any one of (50) to (63) and (66);(70) The pharmaceutical composition according to (69), for treating orpreventing a KLK5-related disease;(71) The pharmaceutical composition according to (70), wherein theKLK5-related disease is Netherton syndrome, atopic dermatitis, rosacea,UV-induced skin injury, psoriasis, asthma, spinal cord injury, cancer,or Barrett's esophagus;(72) The pharmaceutical composition according to any one of (69) to(71), for use in combination with an additional pharmaceutical product;(73) A composition for testing or diagnosis comprising the peptideaccording to any one of (1) to (43) and (49), the polynucleotideaccording to (44), the vector according to (45), the cell according to(46), the conjugate according to any one of (50) to (63) and (66),and/or the antibody or the binding fragment thereof according to (67);(74) The method according to any one of (47), (48), (64), and (65),comprising a step of performing affinity purification using the antibodyor the binding fragment thereof according to (67);(75) A method for identifying a KLK5 inhibitory SPINK2 mutant peptide,comprising the steps (i) to (iii) below: (i) incubating a KLK5 proteaseand a substrate in the presence and absence of a test SPINK2 mutantpeptide; (ii) measuring the protease activity of KLK5 in the presenceand absence of the test SPINK2 mutant peptide; and (iii) determining thepeptide to be positive when the protease activity of KLK5 in thepresence of the peptide is lower than the protease activity of KLK5 inthe absence of the peptide;(76) A method for identifying a KLK5 inhibitory compound, comprising thesteps (i) to (iii) below, using a peptide comprising the amino acidsequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, and 32 (FIGS. 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, and 40) or a conjugate comprising the peptide as areference compound: (i) incubating a KLK5 protease and a substrate inthe presence and absence of a test compound; (ii) measuring the proteaseactivity of KLK5 in the presence and absence of the test compound; and(iii) determining the test compound to be positive when the proteaseactivity of KLK5 in the presence of the test compound is lower than theprotease activity of KLK5 in the absence of the test compound;(77) A method for identifying a KLK5 inhibitory compound, comprising thesteps (i) to (iii) below: (i) measuring a protease inhibitory activityagainst KLK5 of a test compound; (ii) measuring a protease inhibitoryactivity against KLK5 of a reference compound that is a peptidecomprising the amino acid sequence set forth in any one of SEQ ID NOs:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 (FIGS. 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40) or a conjugatecomprising the peptide; and (iii) determining the test compound to bepositive when the protease inhibitory activity against KLK5 of the testcompound is equivalent to or higher than the protease inhibitoryactivity against KLK5 of the reference compound;(78) A method of measuring a KLK5 protease activity, comprising thesteps (i) and (ii) below, using a peptide comprising the amino acidsequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, and 32 (FIGS. 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, and 40) or a conjugate comprising the peptide as areference compound: (i) incubating a KLK5 protease with a substrate, andoptionally another component; and (ii) measuring an amount of thesubstrate and/or an amount of a product after step (i);(79) A KLK5 inhibitory SPINK2 mutant peptide or a SPINK2 mutant peptideconjugate, having a dissociation constant (K_(D)) for KLK5, as measuredby surface plasmon resonance analysis by immobilizing the peptide or theconjugate and adding KLK5 thereto, of 1×10⁻⁹ M or less;(80) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 34 (FIG. 42);(81) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 36 (FIG. 44);(82) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 38 (FIG. 46);(83) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 40 (FIG. 48);(84) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 42 (FIG. 50);(85) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 44 (FIG. 52);(86) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 46 (FIG. 54);(87) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 48 (FIG. 56);(88) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 50 (FIG. 58);(89) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 52 (FIG. 60);(90) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 54 (FIG. 62);(91) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 56 (FIG. 64);(92) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 58 (FIG. 66);(93) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 60 (FIG. 68); and(94) A conjugate comprising the amino acid sequence set forth in SEQ IDNO: 96 (FIG. 106).

Advantageous Effects of Invention

A peptide provided by the present invention, a conjugate containing thepeptide, and a pharmaceutical composition containing the peptide or theconjugate have KLK5 inhibitory activity and are useful for treating orpreventing KLK5-related diseases (which will be described below).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 includes views for comparing the similarities in the amino acidsequences of human KLK5 (SEQ ID NO: 2), KLK7 (SEQ ID NO: 3), and KLK14(SEQ ID NO: 4).

FIG. 2 includes graphs showing the KLK5 inhibitory activity (50%inhibitory concentration: IC₅₀) of each KLK5 inhibitory peptide, usingthe degradation rate of a peptide substrate as an index. For evaluatingthe KLK5 inhibitory activity, KLK5 with a final concentration of 10 nMand Boc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) with a finalconcentration of 100 μM were used.

FIG. 3A includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Bovine trypsin inhibitoryactivity, trypsin (Pierce, 20233) with a final concentration of 5 nM anda substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) witha final concentration of 100 μM were used. For evaluating the Humantrypsin inhibitory activity, trypsin (Sigma-Aldrich Co. LLC, T6424) witha final concentration of 1 nM and a substrate peptideBoc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) with a finalconcentration of 100 μM were used. For evaluating the Bovineα-chymotrypsin inhibitory activity, chymotrypsin (WorthingtonBiochemical Corporation, LS001434) with a final concentration of 10 nMand a substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO: 98)(PEPTIDEINSTITUTE, INC., 3120-v) with a final concentration of 100 μM were used.

FIG. 3B includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human chymotrypsin inhibitoryactivity, chymotrypsin (Sigma-Aldrich Co. LLC, C8946) with a finalconcentration of 10 nM and a substrate peptide Suc-Leu-Leu-Val-Tyr-MCA(SEQ ID NO: 98) (PEPTIDE INSTITUTE, INC., 3120-v) with a finalconcentration of 10 μM were used. For evaluating the Human tryptaseinhibitory activity, tryptase (Sigma-Aldrich Co. LLC, T7063) with afinal concentration of 1 nM and a substrate peptide Boc-Phe-Ser-Arg-MCA(PEPTIDE INSTITUTE, INC., 3107-v) with a final concentration of 100 μMwere used. For evaluating the Human chymase inhibitory activity, chymase(Sigma-Aldrich Co. LLC, C8118) with a final concentration of 100 nM anda substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO: 98) (PEPTIDEINSTITUTE, INC., 3120-v) with a final concentration of 100 μM were used.

FIG. 3C includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human plasmin inhibitoryactivity, plasmin (Sigma-Aldrich Co. LLC, P1867) with a finalconcentration of 50 nM and a substrate peptide Boc-Val-Leu-Lys-MCA(PEPTIDE INSTITUTE, INC., 3104-v) with a final concentration of 100 μMwere used. For evaluating the Human thrombin inhibitory activity,thrombin (Sigma-Aldrich Co. LLC, T6884) with a final concentration of 1nM and a substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems, Inc.,ES011) with a final concentration of 100 μM were used. For evaluatingthe Human neutrophil elastase inhibitory activity, neutrophil elastase(Enzo Life Sciences, BML-SE284) with a final concentration of 0.00001U/μL and a substrate peptide Suc (OMe)-Ala-Ala-Pro-Val-MCA (SEQ ID NO:99)(PEPTIDE INSTITUTE, INC., 3153-v) with a final concentration of 100μM were used.

FIG. 3D includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human matriptase inhibitoryactivity, matriptase (R&D Systems, Inc., 3946-SE) with a finalconcentration of 1 nM and a substrate peptide Boc-Gln-Ala-Arg-AMC (R&DSystems, Inc., ES014) with a final concentration of 100 μM were used.For evaluating the Human protein C inhibitory activity, protein C(Sigma-Aldrich Co. LLC, P2200) with a final concentration of 100 nM anda substrate peptide Boc-Leu-Ser-Thr-Arg-MCA (SEQ ID NO: 100)(PEPTIDEINSTITUTE, INC., 3112-v) with a final concentration of 100 μM were used.For evaluating the Human tPA inhibitory activity, tPA (Sigma-Aldrich Co.LLC, 10831) with a final concentration of 10 nM and a substrate peptidePyr-Gly-Arg-MCA (PEPTIDE INSTITUTE, INC., 3145-v) with a finalconcentration of 100 μM were used.

FIG. 3E includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human uPA inhibitory activity,uPA (Sigma-Aldrich Co. LLC, U0633) with a final concentration of 2 nMand a substrate peptide Pyr-Gly-Arg-MCA (PEPTIDE INSTITUTE, INC.,3145-v) with a final concentration of 100 μM were used. For evaluatingthe Human plasma kallikrein inhibitory activity, plasma kallikrein (R&DSystems, Inc., 2497-SE) with a final concentration of 0.125 μg/mL and asubstrate peptide Z-Phe-Arg-MCA (PEPTIDE INSTITUTE, INC., 3095-v) with afinal concentration of 100 μM were used.

FIG. 3F includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human KLK1 inhibitoryactivity, hKLK1 (R&D Systems, Inc., 2337-SE) with a final concentrationof 0.1 μg/mL and a substrate peptide Pro-Phe-Arg-MCA (PEPTIDE INSTITUTE,INC., 3096-v) with a final concentration of 100 μM were used. Forevaluating the Human KLK2 inhibitory activity, hKLK2 (R&D Systems, Inc.,4104-SE) with a final concentration of 2 μg/mL and a substrate peptidePro-Phe-Arg-MCA (PEPTIDE INSTITUTE, INC., 3096-v) with a finalconcentration of 100 μM were used. For evaluating the Human KLK4inhibitory activity, hKLK4 (R&D Systems, Inc., 1719-SE) with a finalconcentration of 1 μg/mL and a substrate peptide Boc-Val-Pro-Arg-AMC(R&D Systems, Inc., ES011) with a final concentration of 100 μM wereused.

FIG. 3G includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human KLK7 inhibitoryactivity, hKLK7 with a final concentration of 1 μg/mL and a substratepeptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys (Dnp)-NH₂ (SEQ IDNO: 101)(R&D Systems, Inc., ES002) with a final concentration of 20 μMwere used. For evaluating the Human KLK8 inhibitory activity, hKLK8(UniProt: 060259, prepared by the inventors) with a final concentrationof 5 nM and a substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems, Inc.,ES011) with a final concentration of 100 μM were used. For evaluatingthe Human KLK12 inhibitory activity, hKLK12 (R&D Systems, Inc., 3095-SE)with a final concentration of 0.1 μg/mL and a substrate peptideBoc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) with a finalconcentration of 100 μM were used.

FIG. 3H includes graphs to evaluate the cross-reactivity of eachinhibitory peptide with proteases, using the degradation of a peptidesubstrate as an index. For evaluating the Human KLK13 inhibitoryactivity, hKLK13 (R&D Systems, Inc., 2625-SE) with a final concentrationof 0.5 μg/mL, a substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems,Inc., ES011) with a final concentration of 100 μM, and a fluorescencesignal with excitation at 380 nm/emission at 460 nm were used. Forevaluating the Human KLK14 inhibitory activity, hKLK14 with a finalconcentration of 0.2 μg/mL and a substrate peptide Boc-Val-Pro-Arg-AMC(R&D Systems, Inc., ES011) with a final concentration of 100 μM wereused.

FIG. 4 is a view showing a KLK5/KLK5 inhibitory peptide complex obtainedby X-ray crystal structure analysis. The inhibitory peptide K51034 isbound to a region containing the KLK5 active site.

FIG. 5 includes graphs showing the KLK5 inhibitory activity (IC₅₀) ofeach KLK5 inhibitory peptide Fc fusion, using the degradation rate of apeptide substrate as an index. For evaluating the KLK5 inhibitoryactivity, KLK5 with a final concentration of 10 nM andBoc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) with a finalconcentration of 100 μM were used.

FIG. 6A includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Bovine trypsin, Humantrypsin, and Bovine α-chymotrypsin inhibitory activities, the sameconditions as in FIG. 3A were used.

FIG. 6B includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human chymotrypsin,Human tryptase, and Human chymase inhibitory activities, the sameconditions as in FIG. 3B were used.

FIG. 6C includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human plasmin, Humanthrombin, and Human neutrophil elastase inhibitory activities, the sameconditions as in FIG. 3C were used.

FIG. 6D includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human matriptase,Human protein C, and Human tPA inhibitory activities, the sameconditions as in FIG. 3D were used.

FIG. 6E includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human uPA and Humanplasma kallikrein inhibitory activities, the same conditions as in FIG.3E were used.

FIG. 6F includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human KLK1, HumanKLK2, and Human KLK4 inhibitory activities, the same conditions as inFIG. 3F were used.

FIG. 6G includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human KLK7, HumanKLK8, and Human KLK12 inhibitory activities, the same conditions as inFIG. 3G were used.

FIG. 6H includes graphs to evaluate the cross-reactivity of eachinhibitory peptide Fc fusion with proteases, using the degradation of apeptide substrate as an index. For evaluating the Human KLK13 and HumanKLK14 inhibitory activities, the same conditions as in FIG. 3H wereused.

FIG. 7 is a graph showing a KLK5 inhibitory peptide Fcfusion-administered group suppressing the transepidermal water loss(TEWL) in Crusty2 model mice. With hetero (+/−) SPINK5 mutation, skininflammation did not develop, but with homo (+/+) SPINK5 mutation, skininflammation strongly developed, which elevated the TEWL. Administrationof the peptide Fc fusion to the Crusty2 mice with homo (+/+) SPINK5mutation improved dermatitis and reduced the TEWL. The number of casesof Crusty2 (+/−) mice was 9, and the number of cases of Crusty2 (+/+)mice was 12, in each of the PBS-administered group and theD1-K50055-Fc-administered group. The error bars in the figure showstandard errors.

FIG. 8 includes views for comparing the sequence identities in humanSPINK2 (SEQ ID NO: 1), D8 and D9 of human SPINK5 (SEQ ID NO: 88 and SEQID NO: 89, respectively), and human SPINK9 (SEQ ID NO: 90). It is knownthat D8 and D9 of human SPINK5, and human SPINK9 have human KLK5inhibitory activities, but it is seen that the amino acid sequenceidentities with human SPINK2, for which there is no report on human KLK5inhibitory activity, are low in all cases.

FIG. 9 is the amino acid sequence of human SPINK2 (SEQ ID NO: 1).

FIG. 10 is the amino acid sequence of human KLK5 (SEQ ID NO: 2).

FIG. 11 is the amino acid sequence of human KLK7 (SEQ ID NO: 3).

FIG. 12 is the amino acid sequence of human KLK14 (SEQ ID NO: 4).

FIG. 13 is the nucleotide sequence of the KLK5 inhibitory peptide K50032(SEQ ID NO: 5).

FIG. 14 is the amino acid sequence of the KLK5 inhibitory peptide K50032(SEQ ID NO: 6).

FIG. 15 is the nucleotide sequence of the KLK5 inhibitory peptide K50055(SEQ ID NO: 7).

FIG. 16 is the amino acid sequence of the KLK5 inhibitory peptide K50055(SEQ ID NO: 8).

FIG. 17 is the nucleotide sequence of the KLK5 inhibitory peptide K51072(SEQ ID NO: 9).

FIG. 18 is the amino acid sequence of the KLK5 inhibitory peptide K51072(SEQ ID NO: 10).

FIG. 19 is the nucleotide sequence of the KLK5 inhibitory peptide K50016(SEQ ID NO: 11).

FIG. 20 is the amino acid sequence of the KLK5 inhibitory peptide K50016(SEQ ID NO: 12).

FIG. 21 is the nucleotide sequence of the KLK5 inhibitory peptide K51034(SEQ ID NO: 13).

FIG. 22 is the amino acid sequence of the KLK5 inhibitory peptide K51034(SEQ ID NO: 14).

FIG. 23 is the nucleotide sequence of the KLK5 inhibitory peptide K50062(SEQ ID NO: 15).

FIG. 24 is the amino acid sequence of the KLK5 inhibitory peptide K50062(SEQ ID NO: 16).

FIG. 25 is the nucleotide sequence of the KLK5 inhibitory peptide K51090(SEQ ID NO: 17).

FIG. 26 is the amino acid sequence of the KLK5 inhibitory peptide K51090(SEQ ID NO: 18).

FIG. 27 is the nucleotide sequence of the KLK5 inhibitory peptide K50098(SEQ ID NO: 19).

FIG. 28 is the amino acid sequence of the KLK5 inhibitory peptide K50098(SEQ ID NO: 20).

FIG. 29 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideK51028 (SEQ ID NO: 21).

FIG. 30 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideK51028 (SEQ ID NO: 22).

FIG. 31 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideK51005 (SEQ ID NO: 23).

FIG. 32 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideK51005 (SEQ ID NO: 24).

FIG. 33 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideK50031 (SEQ ID NO: 25).

FIG. 34 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideK50031 (SEQ ID NO: 26).

FIG. 35 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideK51057 (SEQ ID NO: 27).

FIG. 36 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideK51057 (SEQ ID NO: 28).

FIG. 37 is the nucleotide sequence of the KLK5/KLK14 inhibitory peptideK51069 (SEQ ID NO: 29).

FIG. 38 is the amino acid sequence of the KLK5/KLK14 inhibitory peptideK51069 (SEQ ID NO: 30).

FIG. 39 is the nucleotide sequence of the KLK5/KLK14 inhibitory peptideK50015 (SEQ ID NO: 31).

FIG. 40 is the amino acid sequence of the KLK5/KLK14 inhibitory peptideK50015 (SEQ ID NO: 32).

FIG. 41 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50032dN-Fc (SEQ ID NO: 33).

FIG. 42 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50032dN-Fc (SEQ ID NO: 34).

FIG. 43 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50055-Fc (SEQ ID NO: 35).

FIG. 44 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50055-Fc (SEQ ID NO: 36).

FIG. 45 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K51072-Fc (SEQ ID NO: 37).

FIG. 46 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K51072-Fc (SEQ ID NO: 38).

FIG. 47 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50016dN-Fc (SEQ ID NO: 39).

FIG. 48 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50016dN-Fc (SEQ ID NO: 40).

FIG. 49 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K51034-Fc (SEQ ID NO: 41).

FIG. 50 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K51034-Fc (SEQ ID NO: 42).

FIG. 51 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50062-Fc (SEQ ID NO: 43).

FIG. 52 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50062-Fc (SEQ ID NO: 44).

FIG. 53 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K51090-Fc (SEQ ID NO: 45).

FIG. 54 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K51090-Fc (SEQ ID NO: 46).

FIG. 55 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50098dN-Fc (SEQ ID NO: 47).

FIG. 56 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50098dN-Fc (SEQ ID NO: 48).

FIG. 57 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51028-Fc (SEQ ID NO: 49).

FIG. 58 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51028-Fc (SEQ ID NO: 50).

FIG. 59 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51005-Fc (SEQ ID NO: 51).

FIG. 60 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51005-Fc (SEQ ID NO: 52).

FIG. 61 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K50031-Fc (SEQ ID NO: 53).

FIG. 62 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K50031-Fc (SEQ ID NO: 54).

FIG. 63 is the nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51057-Fc (SEQ ID NO: 55).

FIG. 64 is the amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51057-Fc (SEQ ID NO: 56).

FIG. 65 is the nucleotide sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K51069dN-Fc (SEQ ID NO: 57).

FIG. 66 is the amino acid sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K51069dN-Fc (SEQ ID NO: 58).

FIG. 67 is the nucleotide sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K50015-Fc (SEQ ID NO: 59).

FIG. 68 is the amino acid sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K50015-Fc (SEQ ID NO: 60).

FIG. 69 is the formula of the SPINK2 mutant peptide (SEQ ID NO: 61).

FIG. 70 is the nucleotide sequence of primer 1 (SEQ ID NO: 62).

FIG. 71 is the nucleotide sequence of primer 2 (SEQ ID NO: 63).

FIG. 72 is the nucleotide sequence of primer 3 (SEQ ID NO: 64).

FIG. 73 is the nucleotide sequence of primer 4 (SEQ ID NO: 65).

FIG. 74 is the nucleotide sequence of primer 5 (SEQ ID NO: 66).

FIG. 75 is the nucleotide sequence of primer 6 (SEQ ID NO: 67).

FIG. 76 is the nucleotide sequence of primer 7 (SEQ ID NO: 68).

FIG. 77 is the nucleotide sequence of primer 8 (SEQ ID NO: 69).

FIG. 78 is the nucleotide sequence of primer 9 (SEQ ID NO: 70).

FIG. 79 is the nucleotide sequence of primer 10 (SEQ ID NO: 71).

FIG. 80 is the nucleotide sequence of primer 11 (SEQ ID NO: 72).

FIG. 81 is the nucleotide sequence of primer 12 (SEQ ID NO: 73).

FIG. 82 is the nucleotide sequence of primer 13 (SEQ ID NO: 74).

FIG. 83 is the nucleotide sequence of primer 14 (SEQ ID NO: 75).

FIG. 84 is the nucleotide sequence of primer 15 (SEQ ID NO: 76).

FIG. 85 is the KLK7 substrate peptide (the amino acid sequence is shownin SEQ ID NO: 77).

FIG. 86 is the bovine α-chymotrypsin substrate peptide (the amino acidsequence is shown in SEQ ID NO: 78).

FIG. 87 is the neutrophil elastase substrate peptide (the amino acidsequence is shown in SEQ ID NO: 79).

FIG. 88 is the human protein C substrate peptide (the amino acidsequence is shown in SEQ ID NO: 80).

FIG. 89 is the nucleotide sequence of primer 16 (SEQ ID NO: 81).

FIG. 90 is the nucleotide sequence of primer 17 (SEQ ID NO: 82).

FIG. 91 is the nucleotide sequence of primer 18 (SEQ ID NO: 83).

FIG. 92 is the nucleotide sequence of primer 19 (SEQ ID NO: 84).

FIG. 93 is the nucleotide sequence of primer 20 (SEQ ID NO: 85).

FIG. 94 is the nucleotide sequence of primer 21 (SEQ ID NO: 86).

FIG. 95 is the amino acid sequence of Fc of human IgG1 (SEQ ID NO: 87).

FIG. 96 is the amino acid sequence of D8 of human SPINKS (SEQ ID NO:88).

FIG. 97 is the amino acid sequence of D9 of human SPINK5 (SEQ ID NO:89).

FIG. 98 is the amino acid sequence of human SPINK9 (SEQ ID NO: 90).

FIG. 99 is the amino acid sequence of mouse KLK5 (SEQ ID NO: 91).

FIG. 100 is the amino acid sequence of mouse KLK7 (SEQ ID NO: 92).

FIG. 101 is the amino acid sequence of mouse KLK14 (SEQ ID NO: 93).

FIG. 102 includes graphs to evaluate the KLK5 inhibitory activity ofeach KLK5 inhibitory peptide Fc fusion (n=3, Mean±SD), using thedegradation rate of a peptide substrate as an index, in order tocalculate inhibition constants K_(i). For evaluating the KLK5 inhibitoryactivity, KLK5 with a final concentration of 10 nM andBoc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) with a finalconcentration of 100 μM were used.

FIG. 103A includes diagrams to evaluate the KLK5 inhibitory activity ofeach KLK5 inhibitory peptide Fc fusion, using the degradation of humanDesmogleinl as an index. For evaluating the KLK5 inhibitory activity,KLK5 with a final concentration of 1 μM and Recombinant HumanDesmoglein-1 Fc Chimera Protein (R&D Systems, Inc., 944-DM-100) with afinal concentration of 1 μM were used. Analysis was performed by Westernblotting using Desmoglein 1 Antibody (aa471-499) (LSBio, LS-C167542) andAnti-Rabbit IgG, HRP-Linked F (ab′) 2 Fragment Donkey (GE healthcare,NA9340V).

FIG. 103B includes diagrams to evaluate the KLK5 inhibitory activity ofeach KLK5 inhibitory peptide Fc fusion, using the degradation of humanDesmocollinl as an index. For evaluating the KLK5 inhibitory activity,KLK5 with a final concentration of 0.2 μM and Recombinant HumanDesmocollin-1 Protein with a C-terminal His tag (R&D Systems, Inc.,4955-DC-050) with a final concentration of 2 μM were used. Analysis wasperformed by Western blotting using Penta His HRP Conjugate (QIAGEN,34460).

FIG. 104 is the nucleotide sequence of primer 22 (SEQ ID NO: 94).

FIG. 105 is the nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D1-K50055-Fc (SEQ ID NO: 95).

FIG. 106 is the amino acid sequence of the KLK5 inhibitory peptide Fcfusion D1-K50055-Fc (SEQ ID NO: 96).

FIG. 107A is a graph showing the KLK5 inhibitory activity (IC₅₀) of theKLK5 inhibitory peptide Fc fusion D1-K50055-Fc, using the degradationrate of a peptide substrate as an index. For evaluating the KLK5inhibitory activity, KLK5 with a final concentration of 10 nM andBoc-Val-Pro-Arg-AMC (R&D Systems, Inc., ES011) with a finalconcentration of 100 μM were used.

FIG. 107B is a graph to evaluate the cross-reactivity of the KLK5inhibitory peptide Fc fusion D1-K50055-Fc with KLK7, using thedegradation rate of a peptide substrate as an index. The same conditionsas in FIG. 3G were used.

FIG. 107C is a graph to evaluate the cross-reactivity of the KLK5inhibitory peptide Fc fusion D1-K50055-Fc with KLK14, using thedegradation rate of a peptide substrate as an index. The same conditionsas in FIG. 3H were used.

DESCRIPTION OF EMBODIMENTS 1. Definition

In the present invention, the term “gene” means a nucleic acid moleculecontaining a nucleotide sequence encoding the amino acid sequence of aprotein or a complementary strand thereof. The meaning of “gene”includes a single-stranded, double-stranded, or triple-stranded or moreassociation of a DNA strand and an RNA strand, and a mixture ofribonucleotides and deoxyribonucleotides on one strand, and double- ortriple- or more stranded nucleic acid molecules containing such strands.

In the present invention, “gene”, “polynucleotide”, and “nucleic acidmolecules” have the same meaning and are not limited at all by thenumber of ribonucleotides, deoxyribonucleotides, nucleotides,nucleosides, and the like, which are their constituent units. Forexample, DNA, RNA, mRNA, cDNA, cRNA, probe, oligonucleotide, primer, andthe like, are also included within the scope of these terms. “Nucleicacid molecules” may be abbreviated as “nucleic acids”.

In the present invention, “polypeptide”, “peptide”, and “protein” havethe same meaning.

In the present invention, a peptide that recognizes target molecule X orbinds to the target molecule X (hereinafter, the recognition or bindingaction will be collectively referred to as “X binding activity”) can bereferred to as an “X binding peptide”. Further, a peptide thatrecognizes the target molecule X or binds to the target molecule X andinhibits or suppresses one or more activities or functions of the targetmolecule X (hereinafter, the inhibitory or suppressive action will becollectively referred to as “X inhibitory activity”) can be referred toas an “X inhibitory peptide”.

In the present invention, “SPINK2” means Serine Protease InhibitorKazal-type 2, which is a 7 kDa protein composed of a Kazal-like domainhaving three disulfide bonds. SPINK2 is preferably derived from humans.In the present invention, human SPINK2 (SEQ ID NO: 1, FIG. 9) will besimply referred to as “SPINK2”, unless otherwise stated.

In the present invention, “KLK5” is a protein composed of an N-terminalpropeptide and a protease active domain and having trypsin-like andchymotrypsin-like protease activities with N-type sugar chains added atthree sites. KLK5 is preferably derived from humans. In the presentinvention, human KLK5 (SEQ ID NO: 2, FIG. 10) may be simply referred toas “KLK5”, unless otherwise stated.

In the present invention, “KLK7” is a protein composed of an N-terminalpropeptide and a trypsin-like domain having a protease activity, with anN-type sugar chain added. KLK7 is preferably derived from humans. In thepresent invention, human KLK7 (SEQ ID NO: 3, FIG. 11) may be simplyreferred to as “KLK7”, unless otherwise stated.

In the present invention, “KLK14” is also called neuropsin and is aprotein composed of an N-terminal propeptide and a trypsin-like domainhaving a protease activity, with an N-type sugar chain added. KLK14 ispreferably derived from humans. In the present invention, human KLK14(SEQ ID NO: 4, FIG. 12) may be simply referred to as “KLK14”, unlessotherwise stated.

In the present invention, “precursor-type KLK5” means pro-KLK5, which iscomposed of a propeptide and a domain having a protease activity.“Active KLK5” means active KLK5, which is composed of a domain having aprotease activity. Active KLK5 is preferably derived from humans.

In the present invention, “precursor-type KLK7” means pro-KLK7, which iscomposed of a propeptide and a domain having a protease activity.“Active KLK7” means active KLK7, which is composed of a domain having aprotease activity. Active KLK7 is preferably derived from humans.

In the present invention, “precursor-type KLK14” means pro-KLK14, whichis composed of a propeptide and a domain having a protease activity.“Active KLK14” means active KLK14, which is composed of a domain havinga protease activity. Active KLK14 is preferably derived from humans.

In the present invention, “KLK5 inhibitory peptide”, “KLK5/KLK7inhibitory peptide”, or “KLK5/KLK14 inhibitory peptide” means a peptidethat inhibits or suppresses one or more activities or functions of KLK5,KLK5 and KLK7, or KLK5 and KLK14, respectively.

The scope of the terms “KLK5 inhibitory peptide”, “KLK5/7 inhibitorypeptide”, and “KLK5/KLK14 inhibitory peptide” include fragments of sucha peptide, and conjugates formed by the addition or binding of othermoieties to the peptide or fragments thereof that maintain the KLK5inhibitory (binding) activity, the KLK5/KLK7 inhibitory (binding)activity, or the KLK5/KLK14 inhibitory (binding) activity, respectively.That is, “KLK5 inhibitory peptide”, “KLK5/KLK7 inhibitory peptide”, or“KLK5/KLK14 inhibitory peptide” includes fragments, adducts, andmodified forms (such as conjugates) of such a peptide that maintain theKLK5 inhibitory (binding) activity, the KLK5 inhibitory (binding)activity and the KLK7 inhibitory (binding) activity, or the KLK5inhibitory (binding) activity and the KLK14 inhibitory (binding)activity, respectively.

In the present invention, “sites” to which the peptide binds, that is,“sites” recognized by the peptide mean consecutive or intermittentpartial amino acid sequences or partial higher-order structures on thetarget molecules to which the peptide binds or recognizes. In thepresent invention, such sites can be called epitopes or binding sites onthe target molecules.

In the present invention, “cells” include various cells derived fromanimal individuals, subcultured cells, primary cultured cells, celllines, recombinant cells, yeasts, and microorganisms.

In the present invention, “SPINK2 mutant” means a peptide containing anamino acid sequence formed by substituting one or more amino acids withamino acids different from those of the wild type, deleting one or morewild-type amino acids, or inserting one or more amino acids that are notfound in the wild type (which will be hereinafter referred tocollectively as “mutation”), in the amino acid sequence of wild-typeSPINK2. “SPINK2 mutants” that have the KLK5 inhibitory activity, theKLK5 inhibitory activity and the KLK7 inhibitory activity (KLK5/KLK7inhibitory activity), or the KLK5 inhibitory activity and the KLK14inhibitory activity (KLK5/KLK14 inhibitory activity) are included withinthe KLK5 inhibitory peptides, KLK5/KLK7 inhibitory peptides, orKLK5/KLK14 inhibitory peptides. In the present invention, “insertion”can be included within the scope of “addition”.

In the present invention, “several” in “one to several” means three toten.

In the present invention, the phrase “hybridizing under stringentconditions” means hybridization in a solution containing 5×SSC at 65° C.and then washing in an aqueous solution containing 2×SSC-0.1% SDS at 65°C. for 20 minutes, in an aqueous solution containing 0.5×SSC-0.1% SDS at65° C. for 20 minutes, and in an aqueous solution containing0.2×SSC-0.1% SDS at 65° C. for 20 minutes, or hybridization underconditions equivalent to the above conditions. SSC means an aqueoussolution of 150 mM NaCl-15 mM sodium citrate, and “n x SSC” means SSC ofn-fold concentration.

In the present invention, the terms “specific” and “specificity” havethe same meaning as “selective” and “selectivity”, respectively, and areinterchangeable. For example, a KLK5-specific inhibitory peptide has thesame meaning as a KLK5-selective inhibitory peptide, and a KLK5- andKLK7-specific inhibitory peptide has the same meaning as a KLK5- andKLK7-selective inhibitory peptide, respectively.

2. Peptide 2-1. Amino Acid

The term “amino acid” means an organic compound containing an aminogroup and a carboxyl group, and preferably means an α-amino acidcontained as a constituent unit in a protein, more preferably in anatural protein. In the present invention, more suitable examples of theamino acid include Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val, and the term“amino acid” means these 20 amino acids in total, unless otherwisenoted. A total of these 20 amino acids can be called “natural aminoacids”. The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide,or the KLK5/KLK14 inhibitory peptide of the present invention preferablycontains a natural amino acid.

In the present invention, “amino acid residues” may be abbreviated as“amino acids”.

Further, in the present invention, the amino acid may be an L-aminoacid, a D-amino acid, or a mixture thereof (DL-amino acid) but meansL-amino acid unless otherwise noted.

The natural amino acids can be classified, for example, into thefollowing groups based on the properties of their common side chains:

(1) Hydrophobic amino acid group: Met, Ala, Val, Leu, and Ile;(2) Neutral hydrophilic amino acid group: Cys, Ser, Thr, Asn, and Gln;(3) Acidic amino acid group: Asp and Glu;(4) Basic amino acid group: His, Lys, and Arg;(5) Group of amino acids that affect the orientation of the main chain:Gly and Pro; and(6) Aromatic amino acid group: Trp, Tyr, and Phe.

However, classification of natural amino acids is not limited to thesegroups.

In the present invention, natural amino acids can undergo a conservativeamino acid substitution.

The term “conservative amino acid substitution” means a substitutionwith a functionally equivalent or similar amino acid. A conservativeamino acid substitution in a peptide results in a static change in theamino acid sequence of the peptide. For example, one or more amino acidshaving the same polarity act functionally equivalently, resulting in astatic change in the amino acid sequence of the peptide. In general, asubstitution within a group can be considered to be conservative instructure and function. However, as will be obvious to those skilled inthe art, the role played by a specific amino acid residue can bedetermined in the context of the three-dimensional structure ofmolecules containing the amino acid. For example, a cysteine residue cantake a less polar oxidized (disulfide) form, as compared with thereduced (thiol) form. A long aliphatic moiety of an arginine side chaincan form a structurally and functionally important feature.

Further, a side chain containing an aromatic ring (such as tryptophan,tyrosine, and phenylalanine) can contribute to ion-aromatic interactionsor cation-pi interactions. In such a case, even if amino acids havingthese side chains are substituted with amino acids belonging to theacidic or non-polar group, they can be structurally and functionallyconservative. Residues such as proline, glycine, and cysteine (disulfideform) can have a direct effect on the three-dimensional structure of themain chain and often cannot be substituted without structuraldistortion.

The conservative amino acid substitution includes specific substitutions(L. Lehninger, Biochemistry, 2^(nd) edition, pp 73-75, Worth Publisher,New York (1975)) and typical substitutions based on the similarity inside chains, as shown below:

(1) Non-polar amino acid group: alanine (which will be hereinafterreferred to as “Ala” or simply “A”), valine (which will be hereinafterreferred to as “Val” or simply “V”), leucine (which will be hereinafterreferred to as “Leu” or simply “L”), isoleucine (which will behereinafter referred to as “Ile” or simply “I”), proline (which will behereinafter referred to as “Pro” or simply “P”), phenylalanine (“Phe” orsimply “F”), tryptophan (which will be hereinafter referred to as “Trp”or simply “W”), and methionine (which will be hereinafter referred to as“Met” or simply “M”);(2) Uncharged polar amino acid group: glycine (which will be hereinafterreferred to as “Gly” or simply “G”), serine (which will be hereinafterreferred to as “Ser” or simply “S”), threonine (which will behereinafter referred to as “Thr” or simply “T”), cysteine (which will behereinafter referred to as “Cys” or simply “C”), tyrosine (which will behereinafter referred to as “Tyr” or simply “Y”), asparagine (which willbe hereinafter referred to as “Asn” or simply “N”), and glutamine (whichwill be hereinafter referred to as “Gln” or simply “Q”);(3) Acidic amino acid group: aspartic acid (which will be hereinafterreferred to as “Asp” or simply “D”) and glutamic acid (which will behereinafter referred to as “Glu” or simply “E”); and(4) Basic amino acid group: lysine (which will be hereinafter referredto as “Lys” or simply “K”), arginine (which will be hereinafter referredto as “Arg” or simply “R”), and histidine (which will be hereinafterreferred to as “His” or simply “H”).

In the present invention, the amino acid may be an amino acid other thana natural amino acid. Examples thereof can include amino acids found innatural peptides or proteins, such as selenocysteine,N-formylmethionine, pyrrolidine, pyroglutamic acid, cystine,hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine, desmosin,β-alanine, sarcosine, ornithine, creatine, γ aminobutyric acid, opine,theanine, tricolominic acid, kainic acid, domoic acid, and acromelicacid, and other amino acids not found in the natural world, such asN-terminal protected amino acids including norleucine, Ac-amino acid,Boc-amino acid, Fmoc-amino acid, Trt-amino acid, and Z-amino acid,C-terminal protected amino acids including amino acid t-butyl ester,benzyl ester, cyclohexyl ester, and fluorenyl ester, diamine, W aminoacid, β amino acid, γ amino acid, Tic derivatives of amino acids, andaminophosphonic acid. However, there is no limitation to these, andamino acids other than the aforementioned 20 “natural amino acids” willbe collectively referred to as “non-natural amino acids” in the presentinvention for convenience of description.

2-2. KLK5 Inhibitory Peptide, KLK5/KLK7 Inhibitory Peptide, andKLK5/KLK14 Inhibitory Peptide

The peptide of the present invention has a KLK5 inhibitory activity, aKLK5/KLK7 inhibitory activity, or a KLK5/KLK14 inhibitory activity.

KLK5, KLK7, and KLK14 targeted by the KLK5 inhibitory peptide, theKLK5/KLK7 inhibitory peptide, and the KLK5/KLK14 inhibitory peptide ofthe present invention are preferably derived from vertebrate animals,more preferably mammals, furthermore preferably primates, optimallyhumans. KLK5, KLK7, and KLK14 can be purified from tissues or cells, orprepared by methods known to those skilled in the art as methods forpreparing proteins such as gene recombination, in-vitro translation, andpeptide synthesis. A signal sequence, an Fc region of an immunoglobulin,a tag, a label, and the like may be linked to KLK5, KLK7, and KLK14. TheKLK5 inhibitory activity, the KLK5/KLK7 inhibitory activity, and theKLK5/KLK14 inhibitory activity can be evaluated using the proteaseactivities of KLK5, KLK5 and KLK7, and KLK5 and KLK14, as indices. Forexample, when KLK5, KLK5 and KLK7, or KLK5 and KLK14, or a functionalfragment thereof is allowed to coexist with a substrate and the KLK5inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14inhibitory peptide of the present invention or a candidate thereof, KLK5inhibition, KLK5/KLK7 inhibition, or KLK5/KLK14 inhibition occurs whenthe protease activity of KLK5, KLK5 and KLK7, or KLK5 and KLK14 is 70%or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less,1% or less, or 0%, as compared with that in the presence of the controlor in the absence of the inhibitor or the candidate thereof. Theinhibitory activity is 30% or more, 50% or more, 70% or more, 80% ormore, 90% or more, 95% or more, 99% or more, or 100%, respectively. TheKLK5 inhibitory activity, the KLK5/KLK7 inhibitory activity, and theKLK5/KLK14 inhibitory activity can vary depending on the reactionconditions, the type of substrate, the concentration, and the like. Thereaction conditions can be exemplified by those described in theExamples, but they are not limited to these examples. The enzymaticactivity can be evaluated by adding a substrate peptide or a substrateprotein to KLK5, KLK5 and KLK7, or KLK5 and KLK14 at a certainconcentration and allowing it to react for a certain time and thereafterdetecting the fluorescence of the substrate peptide or the substrateprotein by SDS-PAGE, Western blotting, liquid chromatography, or thelike. As a buffer, phosphate buffer saline (which will be hereinafterreferred to as “PBS”), tris buffer (50 mM tris, pH 7 to 8.5, forexample, pH 7.5), and the like can be used, and salts such as NaCl (0 to200 mM, for example, 200 mM), CaCl₂) (0 to 10 mM, for example, 2 mM),ZnCl₂, and Brij-35 can be added thereto. However, there is no limitationto these.

The KLK5 inhibitory activity, the KLK5/KLK7 inhibitory activity, and theKLK5/KLK14 inhibitory activity can each be represented by an inhibitionconstant K_(i). The protease activity is measured by adding a substratepeptide to an enzyme at a certain concentration and allowing it to reactfor a certain time and thereafter detecting the fluorescence of thesubstrate peptide. From the protease activity at each substrateconcentration, the maximum reaction rate V_(max) and the Michaelisconstant K_(m) are calculated according to the Michaelis-Menten equation(Michaelis L, et al. (2011) Biochemistry., Vol. 50, No. 39, pp.8264-8269). Further, the inhibition constant K_(i) is calculated fromthe protease activity when each inhibitor is added to an enzyme at acertain concentration according to the Morrison equation (Morrison J F.(1969) Biochim Biophys Acta., Vol. 185, No. 2, pp. 269-286). Examples ofthe software used for the calculation can include GraphPad Prism(GraphPad Software Inc).

The protease substrate of KLK5, KLK7 and KLK5, or KLK5 and KLK14 is notspecifically limited and may be an endogenous substrate, an exogenoussubstrate, a synthetic substrate, or the like. Examples of the humanendogenous substrate of KLK5 can include low-molecular weight kininogenor kallistatin, collagen, Desmoglein, Desmocollin, and Cathelicidin.Examples of the human endogenous substrate of KLK7 can include Pro-KLK5,fibronectin, and collagen. Examples of the human endogenous substrate ofKLK14 can include tPA, fibronectin, and collagen. Gelatin obtained byheat-denaturing collagen can also be used as a substrate. The syntheticsubstrate is not specifically limited, but examples thereof can includePFR-AMC and Boc-VPR-AMC. In the present invention, the KLK5 inhibitoryactivity (IC₅₀ or K_(i)) of the KLK5 inhibitory peptide, the KLK5inhibitory activity of the KLK5/KLK7 inhibitory peptide, and the KLK5inhibitory activity and the KLK14 inhibitory activity of the KLK5/KLK14inhibitory peptide are each 1 μM or less, preferably 300 nM or less,more preferably 100 nM or less, further preferably 30 nM, furthermorepreferably 10 nM or less. The KLK7 inhibitory activity of the KLK5/KLK7inhibitory peptide is preferably 1000 nM or less, more preferably 300 nMor less, further preferably 100 nM, furthermore preferably 30 nM orless. Further, classification of the KLK5/KLK7 inhibitory peptide or theKLK5/KLK14 inhibitory peptide can be made by the relative degree of theKLK5 inhibitory activity and the KLK7 inhibitory activity or the KLK14inhibitory activity (each represented by IC₅₀ or K_(i)). Peptides arepreferably classified into three groups: (i) the KLK5 inhibitoryactivity is less than 0.5 times the KLK7 inhibitory activity or theKLK14 inhibitory activity; (ii) the KLK5 inhibitory activity is morethan 0.5 times and less than 2 times the KLK7 inhibitory activity or theKLK14 inhibitory activity; and (iii) the KLK5 inhibitory activity is 2times or more the KLK7 inhibitory activity or the KLK14 inhibitoryactivity, and desired peptides can be selected from these groupsaccording to their application, such as treatment.

Further, the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide,or the KLK5/KLK14 inhibitory peptide of the present invention preferablydoes not inhibit or suppress protease activities other than KLK5, KLK5and KLK7, or KLK5 and KLK14, respectively, or the degree of inhibitionor suppression against the other protease activities is preferablyrelatively weak. In other words, the protease inhibitory activity of theKLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention preferably hashigh KLK5 specificity, KLK5/KLK7 specificity, or KLK5/KLK14 specificity.The peptide of the present invention preferably does not inhibit orsuppress the activities of proteases such as KLK1, KLK2, KLK3, KLK4,KLK6, KLK8, KLK9 to KLK13, KLK15, chymotrypsin, tryptase, chymase,plasmin, thrombin, elastase, matriptase, protein C, tissue plasminogenactivator (tPA), urokinase plasminogen activator (uPA), and plasmakallikrein, or the degree of inhibition or suppression thereagainst isrelatively weak. Such a preferred peptide of the present invention doesnot exhibit side effects caused by inhibiting or suppressing theactivities of other proteases and is suitable to be used as atherapeutic or prophylactic agent for KLK5-related diseases (which willbe described below). Further, the KLK5-specific inhibitory peptide ofthe present invention preferably does not inhibit or suppress theprotease activities of KLK7 and KLK14, or the degree of inhibition orsuppression thereagainst is relatively weak; the KLK5/KLK7-specificinhibitory peptide of the present invention preferably does not inhibitor suppress the protease activity of KLK14, or the degree of inhibitionor suppression thereagainst is relatively weak; and the KLK5/KLK14inhibitory peptide of the present invention preferably does not inhibitor suppress the protease activities of KLK7, or the degree of inhibitionor suppression thereagainst is relatively weak.

Inhibitors that have low specificity for KLK5, KLK5 and KLK7, or KLK5and KLK14 and inhibit the protease activities of other KLKs in additionto KLK5, KLK5 and KLK7, or KLK5 and KLK14, that is, non-selectiveinhibitors can cause side effects when administered to humans (Coussens,L M, et al., Science, Vol. 295, No. 5564, pp. 2387-2392 (2002); Bissett,D, et al., J. Clin. Oncol., Vol. 23, No. 4, pp. 842-849 (2005)).Meanwhile, inhibitors that have high specificity for KLK5, KLK5/KLK7, orKLK5/KLK14, that is, KLK5-specific inhibitory peptides,KLK5/KLK7-specific inhibitory peptides, or KLK5/KLK14-specificinhibitory peptides can avoid such side effects as described above andtherefore are suitable to be used for treating and preventingKLK5-related diseases (which will be described below).

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention may becompetitive in binding of the protease substrate to KLK5, KLK5 and/orKLK7, or KLK5 and/or KLK14.

As described above, KLK5, KLK7, and KLK14 targeted by the peptide of thepresent invention are derived from vertebrate animals, preferablymammals, more preferably primates, furthermore preferably humans but maybe derived from nonhuman animals, for example, rodents such as rats andmice, and primates such as cynomolgus monkeys, common marmosets, andrhesus monkeys. Peptides with an inhibitory activity against nonhumananimal-derived KLK5, KLK5 and KLK7, or KLK5 and KLK14 can be used, forexample, for diagnosing, testing, treating, or preventing diseasesrelated to KLK5 in nonhuman animals. Further, when such peptides alsoinhibit human KLK5, KLK5 and KLK7, or KLK5 and KLK14, pharmacologicaltests and pharmacokinetic tests using such nonhuman animals as animalpathology models, safety tests and toxicity tests using them as healthyanimals, and the like can be performed in non-clinical research anddevelopment of the peptides as therapeutic or prophylactic agents forKLK5-related diseases (which will be described below).

Further, the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide,and the KLK5/KLK14 inhibitory peptide of the present invention haveadvantages such as low molecular weight, comparatively easy production(which will be described below), excellent physical properties such asstorage stability and thermostability, and a wide range of choices forthe administration route, administration method, formulation, etc., whenused as pharmaceutical compositions (which will be described below), ascompared with other biopolymers such as antibodies used in this field aspharmaceuticals and diagnostic agents. Further, the half-life in bloodwhen used as a pharmaceutical composition can be adjusted to be longerby applying a known method such as the addition of biopolymers andpolymers to increase the molecular weight of the peptide of the presentinvention. The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitorypeptide, and the KLK5/KLK14 inhibitory peptide of the present inventionhave a molecular weight of less than 10,000, preferably less than 8,000,more preferably about 7,000 to 7,200. Further, variable loop moietiescomposed of Cys15 to Cys31 in SEQ ID NO: 61 (FIG. 69) or moietiescomposed of Cys15 to Cys63 (hereinafter, referred to as “moietiescontaining six Cys residues”) having a KLK5 inhibitory activity,KLK5/KLK7 inhibitory activity, or KLK5/KLK14 inhibitory activity arealso included within the KLK5 inhibitory peptide, the KLK5/KLK7inhibitory peptide, or the KLK5/KLK14 inhibitory peptide of the presentinvention, respectively. The variable loop moieties have a molecularweight of less than 2,500, preferably about 1,800 to 2,000, and themoieties containing six Cys have a molecular weight of less than 6,000,preferably about 5,300 to 5,500.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention is a SPINK2mutant (which may be hereinafter abbreviated as “SPINK2 mutant”) inwhich the backbone of SPINK2 is at least partially maintained, andpreferably recognizes a partial peptide, a partial higher-orderstructure, or the like of KLK5, KLK5 and KLK7, or KLK5 and KLK14 orbinds thereto (hereinafter, the recognition or binding action will becollectively referred to as “target binding activity”).

The binding of the SPINK2 mutant to KLK5, KLK7, or KLK14 in the presentinvention can be measured or determined using methods known to thoseskilled in the art such as ELISA, Surface Plasmon Resonance(hereinafter, referred to as “SPR”) analysis, BioLayer Interferometry(hereinafter, referred to as “BLI”), Isothermal Titration calorimetry(hereinafter, referred to as “ITC”), flow cytometry, andimmunoprecipitation.

Examples of ELISA include a method of detecting the KLK5 inhibitorypeptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14 inhibitorypeptide that recognizes and binds to KLK5, KLK5/KLK7, or KLK5/KLK14immobilized on a plate. Antibodies for solid phase that recognize KLK5,KLK5/KLK7, or KLK5/KLK14, or a tag fused to KLK5, KLK5/KLK7, orKLK5/KLK14 can be used for immobilizing KLK5, KLK5/KLK7, or KLK5/KLK14,other than biotin-streptavidin. Labeled antibodies for detection thatrecognize the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide,or the KLK5/KLK14 inhibitory peptide, or a tag fused to the KLK5inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14inhibitory peptide can be used for detecting the KLK5 inhibitorypeptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14 inhibitorypeptide, other than labeled streptavidin. Methods that can be performedfor biochemical analysis such as HRP, alkaline phosphatase, and FITC canbe used for labeling, other than biotin. For detection using an enzymelabel, chromogenic substrates such as TMB(3,3′,5,5′-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolylphosphate), p-NPP (p-nitrophenyl phosphate), OPD (o-Phenylenediamine),ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), and SuperSignal ELISAPico Chemiluminescent Substrate (Thermo Fisher Scientific), fluorescencesubstrates such as QuantaBlu (R) Fluorogenic Peroxidase Substrate(Thermo Fisher Scientific), and chemiluminescence substrates can beused. Absorption plate readers, fluorescence plate readers, luminescenceplate readers, RI liquid scintillation counters, and the like can beused for measuring detection signals.

The measurement method by SPR analysis may be any of: a method ofimmobilizing the SPINK2 mutant peptide on a sensor chip and adding atarget molecule such as KLK5 to measure the binding between the two; anda method of immobilizing a target molecule such as KLK5 on a sensor chipand adding the SPINK2 mutant peptide to measure the binding between thetwo. The former is preferred. For immobilizing the SPINK2 mutantcontained in the peptide of the present invention or the conjugatethereof, a direct method or a capture method can be used. The latter ispreferred. In the direct method, the hydrophobicity of the SPINK2mutant, the amino groups and carboxyl groups of SPINK2, and the like canbe used for direct immobilization. In the capture method, antibodiesthat recognize the conjugate or a tag fused to the conjugate, protein A,protein G, or the like can be used for immobilization, other thanbiotin-streptavidin. A target molecule, such as KLK5 diluted using anassay buffer, is added to the sensor chip on which the SPINK2 mutant isimmobilized, and the SPR signal is observed over time to obtain bindingsensorgrams. Subsequently, an assay buffer, containing no targetmolecules such as KLK5, is added and the SPR signal is observed overtime to obtain dissociation sensorgrams. The binding affinity isanalyzed using the obtained sensorgrams, to calculate a dissociationconstant K_(D). Devices used for the SPR analysis can include BIAcore(R) (GE healthcare), ProteOn (R) (BioRad), SPR-Navi (R) (BioNavisOy),Spreeta (R) (Texas Instruments), SPRi-PlexII (R) (HORIBA, Ltd.), andAutolab SPR (R) (Metrohm). Devices used for BLI can include Octet (R)(Pall).

Examples of immunoprecipitation include a method of detecting KLK5, KLK5and KLK7, or KLK5 and KLK14 recognized by and bound to the KLK5inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14inhibitory peptide immobilized on beads. As regards the beads, magneticbeads, agarose beads, and the like can be used. For immobilizing theKLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide, antibodies that recognize such a peptideor a tag fused to the peptide, protein A, protein G, or the like can beused, other than biotin-streptavidin. The beads are separated by amagnet, centrifugation or the like, and the KLK5, KLK5 and KLK7, or KLK5and KLK14 precipitated together with the beads are detected by SDS-PAGEor Western blotting. For detecting KLK5, KLK5 and KLK7, or KLK5 andKLK14, labeled antibodies for detection that recognize a tag fused toKLK5, KLK7, or KLK14, or KLK5, KLK7, or KLK14 and the like can be used,other than labeled streptavidin. Methods that can be performed forbiochemical analysis such as HRP, alkaline phosphatase, and FITC can beused for labeling, other than biotin. For detection using an enzymelabel, the same substrate as used in ELISA can be used. For measuringdetection signals, ChemiDoc (R) (BioRad), LuminoGraph (ATTO), and thelike can be used.

In the present invention, “specific recognition”, that is, “specificbinding” means binding that is not non-specific adsorption. Examples ofcriteria to determine whether or not the binding is specific can includethe binding activity EC₅₀ in ELISA. Examples of other criteria caninclude the dissociation constant (hereinafter, referred to as “K_(D)”).In the present invention, the K_(D) value of the KLK5 inhibitory peptidefor KLK5, the K_(D) value of the KLK5/KLK7 inhibitory peptide for KLK5and KLK7, or the K_(D) value of the KLK5/KLK14 inhibitory peptide forKLK5 and KLK14 is 1×10⁻⁵ M or less, 5×10⁻⁶ M or less, 2×10⁻⁶ M or less,or 1×10⁻⁶ M or less, more preferably 5×10⁻⁷ M or less, 2×10⁻⁷ M or less,or 1×10⁻⁷ M or less, further preferably 5×10⁻⁸ M or less, 2×10⁻⁸ M orless, or 1×10⁻⁸ M or less, furthermore preferably 5×10⁻⁹ M or less,2×10⁻⁹ M or less, or 1×10⁻⁹ M or less. Examples of other criteria caninclude the analysis results of immunoprecipitation. In the case ofimmobilizing the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitorypeptide, or the KLK5/KLK14 inhibitory peptide on beads, which ispreferred in the present invention, and adding KLK5, KLK5 and KLK7, orKLK5 and KLK14 thereto, followed by separation of the beads, to detectKLK5, KLK5 and KLK7, or KLK5 and KLK14 precipitated together with thebeads, the signals of KLK5, KLK5 and KLK7, or KLK5 and KLK14 aredetected.

The SPINK2 mutant as the KLK5 inhibitory peptide, the KLK5/KLK7inhibitory peptide, or the KLK5/KLK14 inhibitory peptide of the presentinvention can have the protease inhibitory activity, target bindingactivity, other properties, functions, and features as described above,while its full-length amino acid sequence has a high sequence identityto the amino acid sequence of human wild-type SPINK2. The SPINK2 mutantof the present invention has a sequence identity of 60% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more,98% or more or 99% or more, to the amino acid sequence of human SPINK2(SEQ ID NO: 1, FIG. 9).

The term “identity” means a property that indicates the degree ofsimilarity or the relationship between two sequences. The identity (%)of an amino acid sequence is calculated by dividing the number of aminoacids or amino acid residues that are identical by the total number ofamino acids or amino acid residues and multiplying the numerical valueobtained by 100.

The term “gap” means a gap in the alignment between two or moresequences resulting from a deletion and/or addition in at least one ofthe sequences.

The identity between two amino acid sequences that have completelyidentical amino acid sequences is 100%, but when one of the amino acidsequences is compared with the other, and one or more amino acids oramino acid residues are substituted, deleted, or added, the identitybetween the two is less than 100%. Examples of algorithms or programsfor determining the identity between two sequences in consideration of agap can include techniques known to those skilled in the art usingstandard parameters, such as BLAST (Altschul, et al. Nucleic Acids Res.,Vol. 25, pp. 3389-3402, 1997), BLAST2 (Altschul, et al., J. Mol. Biol.,Vol. 215, pp. 403-410, 1990), and Smith-Waterman (Smith, et al., J. Mol.Biol., Vol. 147, pp. 195-197, 1981).

In the present invention, the term “mutated” means that one or morenucleotides, nucleotide residues, amino acids, or amino acid residueshave been substituted, deleted, or inserted in a nucleotide sequence oran amino acid sequence, as compared with the naturally occurring nucleicacid molecule or peptide. The amino acid sequence of the SPINK2 mutantof the present invention has one or more amino acids or amino acidresidues mutated, as compared with the amino acid sequence of humanSPINK2.

In an aspect of the present invention, the amino acid sequence of theSPINK2 mutant may have: one, two, three, four, five, six, or seven aminoacids out of Ser16 to Gly22 substituted with other amino acids or aminoacid residues; one, two, three, four, or five amino acids out of Pro24to Asn28 substituted with other amino acids or amino acid residues; andone, two, three, four, five, or six out of Cys15, Cys23, Cys31, Cys42,Cys45 and Cys63 substituted with other amino acids in order to deletenatural disulfide bonds or to generate non-natural disulfide bonds,although they are preferably the same Cys as in the wild type in orderto maintain the natural disulfide bonds, in the amino acid sequence ofhuman SPINK2 (SEQ ID NO: 1, FIG. 9). In some preferred KLK5 inhibitorypeptides, KLK5/KLK7 inhibitory peptides, or KLK5/KLK14 inhibitorypeptides of the SPINK2 mutant of the present invention, Cys ismaintained at the same 6 sites as in the natural type to retain thedisulfide bonds. In some more preferred aspects of such peptides, Cys15to Cys45, Cys23 to Cys42, and Cys31 to Cys63 each form a disulfide bond.

When the amino acid sequence of such a SPINK2 mutant is contained in theKLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide, a three-dimensional structure constitutedby a loop structure composed of Ser16 to Val30 contained in the aminoacid sequence of the wild-type SPINK2, a β-sheet composed of β strand(1) composed of Cys31 and Gly32 and β strand (2) composed of Ile57 toArg59, and an α-helix composed of Glu41 to Gly51, or a loop structure, aβ-sheet, and an α-helix that are similar to or at least partiallycorrespond to the above (positions thereof) are preferably maintained,to the extent that the KLK5 inhibitory activity, the KLK5/KLK7inhibitory activity, or the KLK5/KLK14 inhibitory activity can beexerted.

The amino acid sequence of the KLK5 inhibitory peptide, the KLK5/KLK7inhibitory peptide, or the KLK5/KLK14 inhibitory peptide according tosome aspects of the SPINK2 mutant of the present invention will bedescribed below. As described above, “amino acid residues” may be simplyexpressed as “amino acids” in the present invention.

In the amino acid sequence (formula) set forth in SEQ ID NO: 61 (FIG.69), X₂ to X₁₂ are not specifically limited, as long as they are anyamino acid such that the resultant mutant inhibits KLK5, KLK5 and KLK7,or KLK5 and KLK14. Hereinafter, suitable amino acids of X₂ to X₁₂ willbe described, but the amino acids may include the same amino acids as inthe amino acid sequence of the natural-type, that is, the wild-typehuman SPINK2.

In the amino acid sequence set forth in SEQ ID NO: 61 (FIG. 69)contained in the KLK5 inhibitory peptide, it is suitable that: Xaa16(X₂) be Ala, Asp, Gly, Gln, Leu, Ser, or Thr; Xaa17 (X₂) be Arg, Glu,Asn, Gln, or Ser; Xaa18 (X₃) be Asp, Gln, Ile, Thr, Trp, or Tyr; Xaa19(X₄) be Arg, Gly, Met, Gln, or Thr; Xaa20 (X₅) be Asp, Glu, Leu, Lys,Thr, or Tyr; Xaa21 (X₆) be Glu, Gly, His, Leu, Ser, Gln, or Tyr; Xaa22(X₇) be Asp, Gly, Gln, Ser, or Tyr; Xaa24 (X₈) be Ala, Asp, Glu, Gly,Asn, Ser, or Thr; Xaa25 (X₉) be Arg or Lys; Xaa26 (X₁₀) be Asp, Glu,Gln, Ser, or Val; Xaa27 (X₁₁) be Phe or Tyr; and Xaa28 (X₁₂) be Asp orGlu.

In the amino acid sequence set forth in SEQ ID NO: 61 (FIG. 69)contained in the KLK5/KLK7 inhibitory peptide, it is suitable that:Xaa16 (X₁) be Gly, Met, or Tyr; Xaa17 (X₂) be Glu, Gln, or Thr; Xaa18(X₃) be His, Met, or Tyr; Xaa19 (X₄) be Ala, Arg, Lys, or Gln; Xaa20(X₅) be Gly, Arg, or Ser; Xaa21 (X₆) be Arg, Lys, Gln, or Ser; Xaa22(X₂) be Gly; Xaa24 (X₈) be His, Thr, or Tyr; Xaa25 (X₉) be His or Tyr;Xaa26 (X₁₂) be Asp, Glu, or His; Xaa27 (X₁₁) be Tyr; and Xaa28 (X₁₂) beAsp or Glu.

In the amino acid sequence set forth in SEQ ID NO: 61 (FIG. 69)contained in the KLK5/KLK14 inhibitory peptide, it is suitable that:Xaa16 (X₁) be Gly, Ser, or Tyr; Xaa17 (X₂) be Asp or Gln; Xaa18 (X₃) beThr or Val; Xaa19 (X₄) be Thr or Val; Xaa20 (X₅) be Glu or Thr; Xaa21(X₆) be His or Thr; Xaa22 (X₂) be Tyr; Xaa24 (X₈) be Asn or Ser; Xaa25(X₉) be Arg; Xaa26 (X₁₀) be Asp or Glu; Xaa27 (X₁₁) be Tyr; and Xaa28(X₁₂) be Asp.

Xaa16 to 22 and Xaa24 to 28 (X₁ to X₁₀) of the wild type arerespectively Ser, Gln, Tyr, Arg, Leu, Pro, Gly, Pro, Arg, His, Phe, andAsn.

In the present invention, one to several or more, preferably 1 to 5amino acids may be further added to the N-terminal side of the firstamino acid. Examples of such an addition of amino acids can preferablyinclude the addition of 1 to 5 Asp and/or Glu (both of Asp and Glu maybe included), more preferably the addition of 1 to 5 Asp or the additionof 1 to 5 Glu.

In the present invention, a peptide that is formed by the substitution,addition, and/or deletion of one or more amino acids in, to or from anadded moiety of an N-terminal and/or C-terminal adduct of the SPINK2mutant peptide (which will be hereinafter referred to as a “parentpeptide”) and partially or fully maintains the activities of the SPINK2mutant peptide may be referred to as a “derivative of the parentpeptide” or a “parent peptide derivative”. Such a “derivative” is alsoincluded within the scope of the “peptide” of the present invention.

The amino acid sequence of the SPINK2 mutant included within the scopeof the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention can containnatural amino acids or mutated amino acids or amino acid sequences inmoieties other than X₁ to X₁₂, that is, at the positions of Pro2 toCys15, Cys23, and Pro29 to Cys63 in the amino acid sequence of wild-typehuman SPINK2 (SEQ ID NO: 1, FIG. 9). For example, the SPINK2 mutant maybe mutated at one or more positions as long as the mutations do notcompletely prevent or interfere with the KLK5 inhibitory activity, theKLK5/KLK7 inhibitory activity, or the KLK5/KLK14 inhibitory activity, orfolding. Such mutations can be achieved using standard methods known tothose skilled in the art. Examples of typical mutations in the aminoacid sequence can include the substitution, deletion or insertion of oneor more amino acids, and examples of a substitution can include aconservative substitution. As a result of a conservative substitution,an amino acid residue is substituted with another amino acid residuehaving similar chemical characteristics not only in bulkiness but alsoin polarity. Examples of conservative substitutions are describedelsewhere in this description. Meanwhile, moieties other than X₁ to X₁₂can allow the non-conservative substitution of one or more amino acids,as long as the substitutions do not completely prevent or interfere withthe KLK5 inhibitory activity, the KLK5/KLK7 inhibitory activity, or theKLK5/KLK14 inhibitory activity, or folding.

In the amino acid sequence of the SPINK2 mutant serving as the KLK5inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14inhibitory peptide of the present invention, X₁ to X₁₂ are preferablyrespectively the amino acids of X₁ to X₁₂ in any one of SEQ ID NOs: 6,8, 10, 12, 14, 16, 18, and 20 (FIGS. 14, 16, 18, 20, 22, 24, 26, and28), SEQ ID NOs: 22, 24, 26, and 28 (FIGS. 30, 32, 34, and 36), and SEQID NOs: 30 and 32 (FIGS. 38 and 40), and moieties other than X₁ to X₁₂can have amino acids or amino acid sequences that do not completelyprevent or interfere with the KLK5 inhibitory activity, the KLK5/KLK7inhibitory activity, or the KLK5/KLK14 inhibitory activity, or folding.

Further, examples of the amino acid sequence of the SPINK2 mutantserving as the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitorypeptide, or the KLK5/KLK14 inhibitory peptide of the present inventioncan include any one of the amino acid sequences (a1) to (a4), (b1) to(b4), or (c1) to (c4) below:

(a1) an amino acid sequence composed of the amino acids at positions 1to 63 in the amino acid sequence set forth in any one of SEQ ID NOs: 6,8, 10, 12, 14, 16, 18, and 20 (FIGS. 14, 16, 18, 20, 22, 24, 26, and28);(a2) an amino acid sequence encoded by a nucleotide sequence thathybridizes with a nucleotide sequence complementary to the nucleotidesequence encoding the amino acid sequence described in (a1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5 inhibitory activity;(a3) an amino acid sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid in the amino acid sequencedescribed in (a1) and contained in a peptide having a KLK5 inhibitoryactivity; and(a4) an amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the amino acid sequencedescribed in (a1) and contained in a peptide having a KLK5 inhibitoryactivity,(b1) an amino acid sequence composed of the amino acids at positions 1to 63 in the amino acid sequence set forth in any one of SEQ ID NOs: 22,24, 26, and 28 (FIGS. 30, 32, 34, and 36);(b2) an amino acid sequence encoded by a nucleotide sequence thathybridizes with a nucleotide sequence complementary to the nucleotidesequence encoding the amino acid sequence described in (b1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5/KLK7 inhibitory activity;(b3) an amino acid sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid in the amino acid sequencedescribed in (b1) and contained in a peptide having a KLK5/KLK7inhibitory activity; and(b4) an amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the amino acid sequencedescribed in (b1) and contained in a peptide having a KLK5/KLK7inhibitory activity, or(c1) an amino acid sequence composed of the amino acids at positions 1to 63 in the amino acid sequence set forth in any one of SEQ ID NOs: 30and 32 (FIGS. 38 and 40);(c2) an amino acid sequence encoded by a nucleotide sequence thathybridizes with a nucleotide sequence complementary to the nucleotidesequence encoding the amino acid sequence described in (c1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5/KLK14 inhibitory activity;(c3) an amino acid sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid in the amino acid sequencedescribed in (c1) and contained in a peptide having a KLK5/KLK14inhibitory activity; and(c4) an amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the amino acid sequencedescribed in (c1) and contained in a peptide having a KLK5/KLK14inhibitory activity.

The amino acids at positions 64 and 65 in SEQ ID NOs: 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, and 32 (FIGS. 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, and 40) are not amino acids corresponding tothose in the wild-type human SPINK2 (SEQ ID NO: 1, FIG. 9: composed of63 amino acids) but are added to express the peptide of the presentinvention in an aspect of the present invention.

Mutations can be introduced into the peptide of the present inventionfor the purpose of improving the folding stability, the thermostability,the storage stability, the half-life in blood, the water solubility, thebiological activity, the pharmacological activity, the side effects, andthe like. For example, new reactive groups such as Cys can be introducedby a mutation for conjugation to other substances such as polyethyleneglycol (PEG), hydroxyethyl starch (HES), biotin, peptides, or proteins.

In the present invention, the KLK5 inhibitory peptide, the KLK5/KLK7inhibitory peptide, or the KLK5/KLK14 inhibitory peptide may be linkedor added to other moieties, and such conjugates are referred to as “KLK5inhibitory peptide conjugates”, “KLK5/KLK7 inhibitory peptideconjugates”, or “KLK5/KLK14 inhibitory peptide conjugates”,respectively. In the present invention, “conjugates” mean moleculesformed by other moieties binding to the peptide of the present inventionor a fragment thereof. The “conjugates” or “conjugation” includemoieties linked or bound to the N-terminus and/or C-terminus of thepeptide of the present invention via a chemical substance such as acrosslinking agent, or via an active substance or the like that issuitable for linking the moieties to an amino acid side chain, by asynthetic chemical method, or by a genetic engineering method or thelike. Example of such “moieties” that improve the half-life in blood caninclude polyalkylene glycol molecules such as polyethylene glycol (PEG),hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid,an Fc region of an immunoglobulin (for example, an Fc region of humanimmunoglobulin G1: its amino acid sequence is shown in SEQ ID NO: 87,FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of animmunoglobulin, albumin or a fragment thereof, albumin binding peptides,albumin binding proteins such as streptococcal protein G, andtransferrin. As regards other “moieties”, the peptide of the presentinvention can be linked to such “moieties” via a linker such as apeptide linker.

In an aspect of the present invention, such a conjugate is a fusion ofthe SPINK2 mutant peptide of the present invention with an Fc region ofan antibody or a fragment thereof. Examples of the origin of theantibody can include humans and nonhuman animals, including rodents suchas mice, rats, and rabbits, other mammals such as bovines, pigs, dogs,cynomolgus monkeys, marmosets, and rhesus monkeys, and birds such aschickens, however, it is preferably from humans.

Examples of the antibody can include IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgD, and IgE, preferably IgG1. More preferably, the conjugate is afusion of the peptide of the present invention with an Fc region ofhuman IgG1 or a fragment thereof. Although the fusion of the peptide ofthe present invention with the Fc region of the antibody or the fragmentthereof may be referred to as “Fc fusion” or “conjugate”, all have thesame meaning.

Examples of the Fc region of human IgG1 can include a region containingor consisting of the amino acid sequence set forth in SEQ ID NO: 87(FIG. 95), but there is no limitation to this. The Fc region of theantibody may be either the wild type or a mutant type.

Further, the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide,or the KLK5/KLK14 inhibitory peptide of the present invention may beconjugated with another drug, in order to exert or enhance thepharmacological activity. Techniques and aspects known to those skilledin the art as antibody-drug conjugates (ADC) in the antibody field canconstitute some aspects of the present invention by replacing theantibody with the peptide of the present invention.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention may furthercontain one or more moieties exerting binding affinity, inhibitoryactivity, antagonist activity, and agonist activity to target moleculesother than KLK5, KLK5 and KLK7, or KLK5 and KLK14, respectively, or maybe conjugated with such moieties. Examples of such “moieties” caninclude antibodies or fragments thereof, and proteins having a backboneother than antibodies such as the SPINK2 mutant or fragments thereof.Techniques and aspects known to those skilled in the art asmultispecific antibodies and bispecific antibodies in the antibody fieldfall within some aspects of the conjugate of the present invention byreplacing at least one of the two or more “antibodies” contained thereinwith the peptide of the present invention.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention, or its precursorcan contain a signal sequence. A signal sequence that is present at theN-terminus of a polypeptide or its precursor or added thereto is usefulfor delivering the polypeptide to a specific compartment of a cell, suchas the periplasm in the case of Escherichia coli and the endoplasmicreticulum in the case of eukaryotic cells. Many signal sequences areknown to those skilled in the art and can be selected according to thehost cells. Examples of the signal sequence for secreting a desiredpeptide into the periplasm of Escherichia coli can include OmpA. Anembodiment containing such a signal sequence can also be included withinthe conjugate of the present invention as an aspect.

Further, the peptide can be purified by affinity chromatography byadding a tag to the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitorypeptide, or the KLK5/KLK14 inhibitory peptide of the present inventionin advance.

The peptide of the present invention, for example, can contain biotin,Strep tag (R), Strep tag II (R), oligohistidines such as His6,polyhistidines, immunoglobulin domains, maltose binding proteins,glutathione-S-transferases (GSTs), calmodulin binding peptides (CBPs),haptens such as digoxigenin and dinitrophenol, epitope tags such as FLAG(R), myc tag, HA tag, and the like (hereinafter, collectively referredto as “affinity tags”) at its C-terminus. The tag adducts can also beincluded within the conjugate of the present invention as some aspects.The conjugate of the present invention, as a whole, may be a peptide(polypeptide).

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention can containlabeling moieties, and can be specifically conjugated with labelingmoieties such as enzyme labels, radioactive labels, colored labels,fluorescence labels, chromogenic labels, luminescent labels, haptens,digoxigenin, biotin, metal complexes, metals, and colloidal gold. Theaspect containing labeling moieties can also be included within theconjugate of the present invention as some aspects.

Examples of the amino acid sequence of the KLK5 inhibitory peptideconjugate, the KLK5/KLK7 inhibitory peptide conjugate, or the KLK5/KLK14inhibitory peptide conjugate of the present invention can include anyone of the amino acid sequences (a1) to (a4), (b1) to (b4), or (c1) to(c4) below:

(a1) the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36,38, 40, 42, 44, 46, 48, and 96 (FIGS. 42, 44, 46, 48, 50, 52, 54, 56,and 106);(a2) an amino acid sequence encoded by a nucleotide sequence thathybridizes with a nucleotide sequence complementary to the nucleotidesequence encoding the amino acid sequence described in (a1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5 inhibitory activity;(a3) an amino acid sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid in the amino acid sequencedescribed in (a1) and contained in a peptide having a KLK5 inhibitoryactivity; and(a4) an amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the amino acid sequencedescribed in (a1) and contained in a peptide having a KLK5 inhibitoryactivity,(b1) the amino acid sequence set forth in any one of SEQ ID NOs: 50, 52,54, and 56 (FIGS. 58, 60, 62, and 64);(b2) an amino acid sequence encoded by a nucleotide sequence thathybridizes with a nucleotide sequence complementary to the nucleotidesequence encoding the amino acid sequence described in (b1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5/KLK7 inhibitory activity;(b3) an amino acid sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid in the amino acid sequencedescribed in (b1) and contained in a peptide having a KLK5/KLK7inhibitory activity; and(b4) an amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the amino acid sequencedescribed in (b1) and contained in a peptide having a KLK5/KLK7inhibitory activity, or(c1) the amino acid sequence set forth in any one of SEQ ID NOs: 58 and60 (FIGS. 66 and 68);(c2) an amino acid sequence encoded by a nucleotide sequence thathybridizes with a nucleotide sequence complementary to the nucleotidesequence encoding the amino acid sequence described in (c1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5/KLK14 inhibitory activity;(c3) an amino acid sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid in the amino acid sequencedescribed in (c1) and contained in a peptide having a KLK5/KLK14inhibitory activity; and(c4) an amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the amino acid sequencedescribed in (c1) and contained in a peptide having a KLK5/KLK14inhibitory activity.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention (the amino acidsequence thereof) can contain both natural amino acids and non-naturalamino acids. The natural amino acids can include both L-amino acids andD-amino acids.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention can exist as amonomer, a dimer, a trimer or higher oligomer, or a multimer. The dimer,the trimer or higher oligomer, and the multimer may be either a homomercomposed of a single type of monomer or a heteromer composed of two ormore different types of monomers. The monomer may rapidly diffuse and beexcellent in penetration into tissues, for example. The dimer, theoligomer, and the multimer can have excellent aspects such as high localaffinity or binding activity to the target molecules, a low dissociationrate, or high KLK5 inhibitory activity, KLK5/KLK7 inhibitory activity,or KLK5/KLK14 inhibitory activity. In addition to spontaneousdimerization, oligomerization, and multimerization, intendeddimerization, oligomerization, and multimerization can also be achievedby introducing a jun-fos domain, a leucine zipper, or the like into thepeptide of the present invention.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide of the present invention can bind to oneor more target molecules or inhibit the activities of the targetmolecules, in the form of a monomer, a dimer, a trimer or higheroligomer, or a multimer.

Examples of the form of the KLK5 inhibitory peptide, the KLK5/KLK7inhibitory peptide, or the KLK5/KLK14 inhibitory peptide of the presentinvention can include isolated forms (such as freeze-dried preparationsand solutions), the aforementioned conjugates, and forms bound to othermolecules (such as immobilized forms, associations with foreignmolecules, and forms bound to target molecules), but there is nolimitation to these examples, and a form suitable for expression,purification, use, storage, or the like can be optionally selected.

3. Identification of KLK5 Inhibitory Peptide, KLK5/KLK7 InhibitoryPeptide, and KLK5/KLK14 Inhibitory Peptide

A KLK5 inhibitory peptide, a KLK5/KLK7 inhibitory peptide, and aKLK5/KLK14 inhibitory peptide can be identified by methods known tothose skilled in the art, using the amino acid sequence of SPINK2 or theamino acid sequence of the KLK5 inhibitory peptide, the KLK5/KLK7inhibitory peptide, and the KLK5/KLK14 inhibitory peptide of the presentinvention (for example, the amino acid sequences according to (a1),(b1), or (c1) above), a nucleotide sequence encoding such an amino acidsequence, nucleic acid molecules containing such a nucleotide sequence,and the like as starting materials. As a suitable example, suchidentification can be made from a human SPINK2 mutant library, using theKLK5 inhibitory activity, the KLK5/KLK7 inhibitory activity, or theKLK5/KLK14 inhibitory activity, respectively, as an index, and thebinding activity to KLK5, KLK5/KLK7, or KLK5/KLK14 may be combined foruse, respectively, as an index.

For example, the nucleic acid molecules serving as the starting materialcan be subjected to mutagenesis and introduced into an appropriatebacterial or eukaryotic host using recombinant DNA technology. TheSPINK2 mutant library is known as a technique for identifying a binderor an inhibitor of a target molecule. For example, the disclosure inInternational Publication No. WO 2014/024914 is also incorporated withinthe disclosure of the present invention by reference in its entirety.After the nucleotide sequence subjected to mutagenesis is expressed inthe appropriate host, a clone formed by linking the SPINK2 mutant havingthe desired properties, activities, functions, and the like with itsgenetic trait can be concentrated and/or selected from the library to beidentified. For concentrating and/or selecting the clone, methods knownto those skilled in the art can be used, such as the bacterial displaymethod (Francisco, J. A., et al. (1993) Proc. Natl. Acad. Sci., USA,Vol. 90, pp. 10444-10448), the yeast display method (Boder, E. T., etal. (1997) Nat. Biotechnol., Vol. 15, pp. 553-557), the mammalian celldisplay method (Ho M, et al. (2009) Methods Mol Biol., Vol. 525, pp.337-352), the phage display method (Smith, G. P. (1985) Science., Vol.228, pp. 1315-1317), the ribosome display method (Mattheakis L C, et al.(1994) Proc. Natl. Acad. Sci., USA, Vol. 91, No. 19, pp. 9022-9029), thenucleic acid display method (Nemoto N, et al. (1997) FEBS Lett., Vol.414, No. 2, pp. 405-408) such as mRNA display, and the colony screeningmethod (Pini, A. et al. (2002) Comb. Chem. High Throughput Screen., Vol.5, pp. 503-510). The nucleotide sequence of the SPINK2 mutant containedin the clone thus selected and identified is determined. Thereby, anamino acid sequence encoded by the nucleotide sequence can be determinedas the amino acid sequence of the SPINK2 mutant, that is, the KLK5inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14inhibitory peptide contained in the clone.

The SPINK2 mutant of the present invention can be obtained, for example,by mutagenesis of the natural SPINK2. “Mutagenesis” means enablingsubstitution or deletion of one or more amino acids present at somepositions in an amino acid sequence with other amino acids, or insertionof amino acids that are not present in the amino acid sequence. Suchdeletion or insertion can change the sequence length. In the SPINK2mutant of the present invention, mutagenesis can preferably occur at oneor more positions of X₁ to X₁₂ in the amino acid sequence set forth inSEQ ID NO: 61 (FIG. 69).

However, the scope of the mutant also includes those with the same aminoacids as present at specific positions in the natural amino acidsequence, that is, a natural amino acid sequence is maintained at one ormore positions of X₁ to X₁₂ after such suitable mutagenesis, as long asat least one amino acid is mutated overall. Likewise, in an aspect ofthe present invention, the scope of the mutant also includes those withthe same amino acids as present at specific positions in the naturalamino acid sequence, that is, a natural amino acid sequence ismaintained at one or more positions in moieties other than X₁ to X₁₂after mutation is induced at the positions, as long as at least oneamino acid is mutated overall.

The term “random mutagenesis” means that one or more different aminoacids are introduced at specific positions within a sequence bymutagenesis with a certain probability, but not all the probabilities ofintroducing the amino acids may be the same. Further, in the presentinvention, the inclusion of the naturally-occurring amino acid (as oneof the amino acids) in the at least two different amino acids cannot beprevented, and the scope of “random mutagenesis” also includes such acase.

As regards a method for random mutagenesis at specific positions,standard methods known to those skilled in the art can be used. Forexample, mutations can be induced at specific positions in a sequence byPCR (polymerase chain reaction) using a mixture of syntheticoligonucleotides containing a degenerate nucleotide composition. Forexample, use of a codon NNK or NNS (N=adenine, guanine, cytosine, orthymine; K=guanine or thymine; S=adenine or cytosine) induces a mutationto introduce a stop codon in addition to all 20 natural amino acids,whereas use of a codon VVS (V=adenine, guanine, or cytosine) eliminatesthe possibility of introducing Cys, Ile, Leu, Met, Phe, Trp, Tyr, andVal and induces a mutation to introduce the other 12 natural aminoacids. Further, use of a codon NMS (M=adenine or cytosine) eliminatesthe possibility of introducing Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp,and Val and induces a mutation to introduce the other 11 natural aminoacids, for example. Special codons, artificial codons, or the like canbe used to induce a mutation to introduce non-natural amino acids.

Site-specific mutagenesis can also be performed using structuralinformation containing a higher-order structure of a target and/or apeptide against the target or a wild-type peptide from which the peptideis derived. In the present invention, site-specific mutation can beintroduced using structural information containing higher-orderinformation on the target KLK5, KLK7, or KLK14, and/or the SPINK2mutant, the wild-type SPINK2 or a complex of the two, with respect toKLK5, KLK5 and KLK7, or KLK5 and KLK14. There may cases where acorrelation can be found between the KLK5 inhibitory activity, theKLK5/KLK7 inhibitory activity, or the KLK5/KLK14 inhibitory activity andthe structural information obtained, for example, through identifying aSPINK2 mutant having the KLK5 inhibitory activity, the KLK5/KLK7inhibitory activity, or the KLK5/KLK14 inhibitory activity, thenobtaining crystals of KLK5, KLK7, or KLK14 and the SPINK2 mutant complexto perform X-ray crystal structure analysis, and specifying an epitopeon the KLK5, KLK7, or KLK14 molecule to which the SPINK2 mutant bindsand a paratope on the SPINK2 mutant corresponding to the epitope basedon the analysis results. Based on the structure-activity correlation,substitution with specific amino acids at specific positions, orinsertion or deletion of amino acids at specific positions or the likeis designed, so that the KLK5 inhibitory activity, the KLK5/KLK7inhibitory activity, or the KLK5/KLK14 inhibitory activity can beactually confirmed.

Further, mutations can be induced, for example, using a nucleotideconstituent unit that modifies the specificity of a base pair, such asinosine.

Further, mutations can be induced at random positions, for example, bythe error-prone PCR method using a DNA polymerase with a high error ratewithout a proofreading function, such as Taq DNA polymerase, or chemicalmutagenesis, and the like.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide can be concentrated and/or selected fromsuitable libraries, such as a phage library and a colony library, knownto those skilled in the art for the respective screening method usingbacterial display, yeast display, mammalian cell display, phage display,ribosome display, nucleic acid display, colony screening, or the like.Using vectors and methods known to those skilled in the art that aresuitable for the respective libraries, such as a phagemid for the phagelibrary and a cosmid for colony screening in these libraries,construction of the libraries can be achieved. The vectors may beviruses that infect prokaryotic or eukaryotic cells or viral vectors.Such recombinant vectors can be prepared by methods known to thoseskilled in the art such as genetic engineering.

Bacterial display is, for example, a technique of fusing a desiredprotein with a part of the outer membrane lipoprotein (Lpp) ofEscherichia coli and the outer membrane protein OmpA to present thedesired protein on the surface of Escherichia coli. A DNA group obtainedby random mutagenesis of a nucleotide sequence encoding an amino acidsequence of a protein is introduced into vectors suitable for bacterialdisplay to transform bacterial cells with the vectors. Thus, a librarypresenting a randomly mutagenized protein group can be obtained on thesurface of the transformed bacterial cells (Francisco, J. A., et al.(1993) Proc. Natl. Acad. Sci., USA, Vol. 90, pp. 10444-10448).

Yeast display is a technique of fusing a desired protein with a proteinsuch as α-agglutinin on the outer shell of the surface of a yeast cellto present it on the yeast surface. The α-agglutinin contains aC-terminal hydrophobic region, which is thought to be aglycosylphosphatidylinositol (GPI) anchor attachment signal, a signalsequence, an active domain, a cell wall domain, and the like, and thedesired protein can be displayed on the surface of the yeast cell bymanipulation of them. A DNA group obtained by random mutagenesis of anucleotide sequence encoding an amino acid sequence of a protein isintroduced into vectors suitable for yeast display to transform yeastcells with the vectors. Thus, a library presenting a randomlymutagenized protein group can be obtained on the surface of thetransformed yeast cells (Ueda, M. & Tanaka, A., Biotechnol. Adv., Vol.18, pp. from 121, Published in 2000; Ueda, M. & Tanaka, A., J. Biosci.Bioeng., Vol. 90, pp. from 125, Published in 2000).

Animal cell display is, for example, a technique of fusing a desiredprotein with a transmembrane region of a membrane protein typified by aplatelet-derived growth factor receptor (PDGFR) to present the desiredprotein on the surface of a mammalian cell, such as HEK293 and Chinesehamster ovary (CHO) cells. A DNA group obtained by random mutagenesis ofa nucleotide sequence encoding an amino acid sequence of a protein isintroduced into vectors suitable for animal cell display to transfectanimal cells with the vectors. Thus, a library presenting a randomlymutagenized protein group can be obtained on the surface of thetransfected animal cells (Ho M, et al. (2009) Methods Mol Biol., Vol.525, pp. 337-352).

The desired library presented on cells such as yeast, bacteria, andanimal cells can be incubated in the presence of the target molecules orbrought into contact with the target molecules. For example, after acell containing the library and KLK5, KLK7, or KLK14 modified withbiotin or the like are incubated for a certain time, a carrier such asmagnetic beads is added thereto, the cell is separated from the carrier,and then the carrier is washed to remove non-specific adsorbates andbinders. Thus, a cell group presenting a peptide bound to the carrier(or KLK5, KLK7, or KLK14 bound to the carrier), an assembly of thepeptides, or the concentrated peptide assembly can be collected.Likewise, a cell group presenting a peptide bound to the carrier (orKLK5, KLK7, or KLK14 bound to the carrier) or KLK5, KLK7, or KLK14, anassembly of the peptides, or the concentrated peptide assembly can becollected by performing magnetic cell separation (MACS) after additionof the magnetic beads or FACS after cell staining using an anti-KLK5antibody, an anti-KLK7 antibody, or an anti-KLK14 antibody. Non-specificadsorbate sites and/or binding sites can be subjected, for example, toblocking treatment, and a blocking step can be incorporated as long asit is an appropriate method. A vector expressing the peptide, thepeptide assembly, or the concentrated peptide assembly thus obtained iscollected, and a nucleotide sequence of the polynucleotide inserted intothe vector is determined, so that an amino acid sequence encoded by thenucleotide sequence can be determined. Further, the peptide assemblybinding to the target molecules can be more highly concentrated byintroducing the vector into the host cell again and repeating theaforementioned operation as a cycle once to several times.

In the case of phage display, a phagemid is, for example, a bacterialplasmid containing a second origin of replication derived from asingle-stranded bacteriophage other than the origin of plasmidreplication. A cell containing a phagemid can replicate the phagemid viaa single-stranded replication mode by superinfection with M13 or asimilar helper bacteriophage. That is, single-stranded phagemid DNA ispackaged in infectious particles coated with a bacteriophage coatprotein. Thus, phagemid DNA can be formed in infected bacteria as acloned double-stranded DNA plasmid, and a phagemid can be formed asbacteriophage-like particles from the culture supernatant of thesuperinfected cell. The particles themselves can be reformed as plasmidsby injecting the bacteriophage-like particles into bacteria having anF-pilus in order to infect the bacteria with such DNA.

A fusion gene containing a polynucleotide having a nucleotide sequenceencoding the amino acid sequence of the test peptide and a bacteriophagecoat protein gene is inserted into the phagemid to infect the bacteria,and such cells are cultured. Thus, the peptide can be expressed orpresented (the same meaning as displayed) on the bacteria or thephage-like particles or can be produced as a fusion protein with thecoat protein in the phage particles or the culture supernatant of thebacteria.

For example, the peptide can be produced in the culture supernatant ofEscherichia coli as a fusion protein containing the peptide and the coatprotein by inserting a fusion gene containing the polynucleotide and thebacteriophage coat protein gene gpIII into a phagemid for superinfectionof Escherichia coli with M13 or a similar helper phage.

In the case of using various cyclic or non-cyclic vectors such as aviral vector instead of the phagemid, a peptide having an amino acidsequence encoded by the nucleotide sequence of the polynucleotideinserted into such a vector can be expressed or presented on the cell inwhich the vector is introduced or on virus-like particles, or can beproduced in the culture supernatant of the cell according to methodsknown to those skilled in the art.

The library expressing the peptide thus obtained can be incubated in thepresence of the target molecules or brought into contact with the targetmolecules. For example, a carrier to which KLK5, KLK5 and/or KLK7, orKLK5 and/or KLK14 is immobilized is incubated for a certain timetogether with a mobile phase containing the library, thereafter themobile phase is separated from the carrier, and then the carrier iswashed to remove non-specific adsorbates and binders. Thus, a peptidebound to the carrier (or KLK5, KLK5 and/or KLK7, or KLK5 and/or KLK14bound to the carrier), an assembly of the peptides, or the concentratedpeptide assembly can be collected by elution. The elution can benon-selectively performed under relatively high ionic strength, low pH,and moderate denaturing conditions, in the presence of chaotropic salts,and the like, or can be selectively performed by adding soluble targetmolecules such as KLK5, KLK7, and KLK14, antibodies that bind to thetarget molecules, natural ligands, substrates, and the like to allow themixture to compete with the immobilized target molecules. Non-specificadsorbate sites and/or binding sites can be subjected, for example, toblocking treatment, and a blocking step can be incorporated as long asit is an appropriate method.

A vector expressing the peptide, the peptide assembly, or theconcentrated peptide assembly thus obtained is collected, and anucleotide sequence of the polynucleotide inserted into the vector isdetermined, so that an amino acid sequence encoded by the nucleotidesequence can be determined. Further, the peptide assembly binding to thetarget molecules can be more highly concentrated by introducing thevector into the host cell again and repeating the aforementionedoperation as a cycle once to several times.

Ribosome display is a technique of synthesizing a desired protein andmRNA corresponding thereto, and a ribosome-linked molecule within a testtube, for example, using mRNA encoding the desired protein free from atermination codon and a cell-free protein synthesis system. A librarypresenting a randomly mutagenized protein group on ribosomes can beobtained, using the mRNA group obtained by random mutagenesis of anucleotide sequence encoding an amino acid sequence of a protein and acell-free protein synthesis system (Mattheakis L C, et al. (1994) Proc.Natl. Acad. Sci., USA, Vol. 91, No. 19, pp. 9022-9029).

Nucleic acid display is also called mRNA display and is a technique ofsynthesizing a desired protein, mRNA encoding the protein, and aribosome-linked molecule, for example, using a linker such as puromycinwhich has a similar structure to the 3′ end of tyrosyl tRNA. Since sucha technique uses a cell-free protein synthesis system instead of livingcells, synthesis within a test tube is possible. A library presenting arandomly mutagenized protein group on ribosomes can be obtained, usingan mRNA group obtained by random mutagenesis of a nucleotide sequenceencoding an amino acid sequence of a protein, a linker such aspuromycin, and a cell-free protein synthesis system (Nemoto N, et al.(1997) FEBS Lett., Vol. 414, No. 2, pp. 405-408).

The library obtained through a cell-free synthesis system such asribosome display or nucleic acid display and expressing the peptide canbe incubated in the presence of the target molecules or brought intocontact with the target molecules. For example, a carrier to which KLK5,KLK5 and/or KLK7, or KLK5 and/or KLK14 is immobilized is incubated for acertain time together with a mobile phase containing the library,thereafter the mobile phase is separated from the carrier, and then thecarrier is washed to remove non-specific adsorbates and binders. Thus, apeptide bound to the carrier (or KLK5, KLK5 and/or KLK7, or KLK5 and/orKLK14 bound to the carrier), an assembly of the peptides, or theconcentrated peptide assembly can be collected by elution. The elutioncan be non-selectively performed under relatively high ionic strength,low pH, and moderate denaturing conditions, in the presence ofchaotropic salts, and the like, or can be selectively performed byadding soluble target molecules such as KLK5, KLK7, and KLK14,antibodies that bind to the target molecules, natural ligands,substrates, and the like to allow the mixture to compete with theimmobilized target molecules. Non-specific adsorbate sites and/orbinding sites can be subjected, for example, to blocking treatment, anda blocking step can be incorporated as long as it is an appropriatemethod.

Nucleic acids expressing the peptide, the peptide assembly, or theconcentrated peptide assembly thus obtained are collected, and thenucleotide sequence is determined after reverse transcription to cDNA inthe case of mRNA, so that an amino acid sequence encoded by thenucleotide sequence can be determined. Further, the peptide assemblybinding to the target molecules can be more highly concentrated bytranscribing mRNA from the nucleic acids thus collected and repeatingthe aforementioned operation as a cycle once to several times.

The peptide or its assembly can be efficiently purified by conjugatingan affinity tag to the peptide, the peptide assembly, or theconcentrated peptide assembly in advance. For example, the peptide canbe eluted by conjugating a substrate of a protease as a tag to thepeptide assembly in advance and then cleaving it through the proteaseactivity.

Based on the sequence information obtained and the functions or the likeof the peptide, further mutations are induced in the clone or thelibrary obtained, so that a peptide with functions (for example, theKLK5 inhibitory activity, the KLK5/KLK7 inhibitory activity, or theKLK5/KLK14 inhibitory activity), physical properties (such asthermostability and storage stability), pharmacokinetics (such asdistribution and half-life in blood), and the like which are improvedcan be obtained from the library to which the mutation has beenintroduced.

The KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide can be identified by determining whetheror not the obtained peptide has the KLK5 inhibitory activity, theKLK5/KLK7 inhibitory activity, or the KLK5/KLK14 inhibitory activity,respectively.

Further, the KLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide,or the KLK5/KLK14 inhibitory peptide can preferably maintain athree-dimensional structure constituted by a loop structure composed ofSer16 to Val30 contained in the amino acid sequence of the wild-typeSPINK2, a β-sheet composed of β strand (1) composed of Cys31 and Gly32and β strand (2) composed of Ile57 to Arg59, and an α-helix composed ofGlu41 to Gly51, or a loop structure, a β-sheet, and an α-helix that aresimilar to or at least partially correspond to the above (positionsthereof), to the extent that the KLK5 inhibitory activity, the KLK5/KLK7inhibitory activity, or the KLK5/KLK14 inhibitory activity can beexerted. It is also possible to identify a more suitable KLK5 inhibitorypeptide, KLK5/KLK7 inhibitory peptide, or KLK5/KLK14 inhibitory peptideusing such a three-dimensional structure (the entire structure or apartial structure) as a part of the index.

Further, the present invention relates to a method for identifying aKLK5 inhibitory peptide, a KLK5/KLK7 inhibitory peptide, or a KLK5/KLK14inhibitory peptide using a SPINK2 mutant library, and examples thereofcan include the method described in (75) above. Further, the presentinvention relates to a method for identifying a KLK5 inhibitorycompound, a KLK5/KLK7 inhibitory compound, or a KLK5/KLK14 inhibitorycompound, using various compound libraries. In such a method, thepeptide of the present invention or the conjugate thereof can be used asa reference compound, a control, or the like, and such a method can beexemplified by (76) above. In such a method, a test compound may bedetermined to be positive when the enzyme inhibitory activity of thecompound is equivalent to or stronger than the enzyme inhibitoryactivity of the reference compound or the control, whereas the compoundmay be determined to be negative when the enzyme inhibitory activity isweaker. A method involving such a comparison can be exemplified by (77)above. Meanwhile, the peptide of the present invention or the conjugatethereof can be used as a reference compound, a control, or the like, inany test involving a step of measuring the protease activity of KLK5 andoptionally KLK7 or KLK14, and such a test method is also included in thepresent invention. Such a test is not specifically limited and can beexemplified by (78) above. Suitable examples thereof can include adiagnostic method, a test method, a detection method, and a method foridentifying an individual to whom a pharmaceutical composition is to beadministered of the present invention (all will be described below).

4. Nucleic Acid Molecules Encoding Peptide of Present Invention orConjugate Thereof, Vector Containing the Same, Cell Containing the Same,and Method for Producing Recombinant Peptide or Conjugate

The present invention also provides a polynucleotide containing thenucleotide sequence encoding the amino acid sequence contained in theKLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide (which will be hereinafter referred to as“nucleic acid molecules encoding the KLK5 inhibitory peptide”, “nucleicacid molecules encoding the KLK5/KLK7 inhibitory peptide”, or “nucleicacid molecules encoding the KLK5/KLK14 inhibitory peptide”), arecombinant vector with such a gene inserted, a cell with the gene orthe vector introduced (which will be hereinafter referred to as a “cellcontaining nucleic acid molecules encoding the KLK5 inhibitory peptide”,a “cell containing nucleic acid molecules encoding the KLK5/KLK7inhibitory peptide”, or a “cell containing nucleic acid moleculesencoding the KLK5/KLK14 inhibitory peptide”), a cell that produces theKLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide (which will be hereinafter referred to asa “cell producing the KLK5 inhibitory peptide”, a “cell producing theKLK5/KLK7 inhibitory peptide”, or a “cell producing the KLK5/KLK14inhibitory peptide”).

Suitable examples of the nucleic acid molecules encoding the KLK5inhibitory peptide, the nucleic acid molecules encoding the KLK5/KLK7inhibitory peptide, or the nucleic acid molecules encoding theKLK5/KLK14 inhibitory peptide of the present invention can include thosecontaining the nucleotide sequence described in any one of (a1) to (a4),(b1) to (b4), or (c1) to (c4) below (which will be hereinafter referredto as the “nucleotide sequence of the KLK5 inhibitory peptide”, the“nucleotide sequence of the KLK5/KLK7 inhibitory peptide”, or the“nucleotide sequence of the KLK5/KLK14 inhibitory peptide”,respectively), those composed of a nucleotide sequence containing thenucleotide sequence of the KLK5 inhibitory peptide, the nucleotidesequence of the KLK5/KLK7 inhibitory peptide, or the nucleotide sequenceof the KLK5/KLK14 inhibitory peptide, or those composed of thenucleotide sequence of the KLK5 inhibitory peptide, the nucleotidesequence of the KLK5/KLK7 inhibitory peptide, or the nucleotide sequenceof the KLK5/KLK14 inhibitory peptide:

(a1) a nucleotide sequence composed of nucleotides 1 to 189 in anucleotide sequence encoding an amino acid sequence composed of theamino acids at positions 1 to 63 in the amino acid sequence set forth inany one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20 (FIGS. 14, 16,18, 20, 22, 24, 26, and 28) or the nucleotide sequence described in anyone of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, and 19 (FIGS. 13, 15, 17,19, 21, 23, 25, and 27);(a2) a nucleotide sequence that hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence described in (a1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5 inhibitory activity;(a3) a nucleotide sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue in thenucleotide sequence described in (a1) and encodes the amino acidsequence contained in a peptide having a KLK5 inhibitory activity; and(a4) a nucleotide sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the nucleotide sequencedescribed in (a1) and encodes the amino acid sequence contained in apeptide having a KLK5 inhibitory activity:(b1) a nucleotide sequence encoding an amino acid sequence composed ofthe amino acids at positions 1 to 63 in the amino acid sequence setforth in any one of SEQ ID NOs: 22, 24, 26, and 28 (FIGS. 30, 32, 34,and 36) or a nucleotide sequence composed of nucleotides 1 to 189 in thenucleotide sequence described in any one of SEQ ID NOs: 21, 23, 25, and27 (FIGS. 29, 31, 33, and 35);(b2) a nucleotide sequence that hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence described in (b1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5/KLK7 inhibitory activity;(b3) a nucleotide sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue in thenucleotide sequence described in (b1) and encodes the amino acidsequence contained in a peptide having a KLK5/KLK7 inhibitory activity;and(b4) a nucleotide sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the nucleotide sequencedescribed in (b1) and encodes the amino acid sequence contained in apeptide having a KLK5/KLK7 inhibitory activity:(c1) a nucleotide sequence encoding an amino acid sequence composed ofthe amino acids at positions 1 to 63 in the amino acid sequence setforth in any one of SEQ ID NOs: 30 and 32 (FIGS. 38 and 40), or anucleotide sequence composed of nucleotides 1 to 189 in the nucleotidesequence described in SEQ ID NO: 29 or 31 (FIG. 37 or 39);(c2) a nucleotide sequence that hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence described in (c1) understringent conditions and encodes the amino acid sequence contained in apeptide having a KLK5/KLK14 inhibitory activity;(c3) a nucleotide sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue in thenucleotide sequence described in (c1) and encodes the amino acidsequence contained in a peptide having a KLK5/KLK14 inhibitory activity;and(c4) a nucleotide sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the nucleotide sequencedescribed in (c1) and encodes the amino acid sequence contained in apeptide having a KLK5/KLK14 inhibitory activity.

A SPINK2 mutant peptide composed of an amino acid sequence encoded bythe nucleotide sequence described in any one of (a1) to (a4), (b1) to(b4), or (c1) to (c4) above or containing an amino acid sequence thatinhibits the protease activity of KLK5, KLK5 and KLK7, or KLK5 andKLK14, preferably that specifically inhibits the protease activitythereof.

Nucleotides 190 to 195 in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, and 31 (FIGS. 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, and 39) are not nucleotides corresponding to those in thenucleotide sequence encoding the wild-type human SPINK2 (SEQ ID NO: 1,FIG. 9: composed of 63 amino acids) but are added in order to expressthe peptide of the present invention in an aspect of the presentinvention.

However, the nucleic acid molecules encoding the KLK5 inhibitorypeptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14 inhibitorypeptide are not limited to those described in (a1) to (a4), (b1) to(b4), or (c1) to (c4), and nucleic acid molecules containing anucleotide sequence encoding an amino acid sequence contained in aSPINK2 mutant having a KLK5 inhibitory activity, KLK5/KLK7 inhibitoryactivity, or KLK5/KLK14 inhibitory activity, preferably, having theamino acid sequence set forth in SEQ ID NO: 61 (FIG. 69) are allincluded within the scope of the nucleic acid molecules encoding theKLK5 inhibitory peptide, the KLK5/KLK7 inhibitory peptide, or theKLK5/KLK14 inhibitory peptide.

Further, the present invention also provides a polynucleotide containingthe nucleotide sequence encoding the amino acid sequence contained in aKLK5 inhibitory peptide conjugate, a KLK5/KLK7 inhibitory peptideconjugate, or a KLK5/KLK14 inhibitory peptide conjugate (which will behereinafter referred to as “nucleic acid molecules encoding the KLK5inhibitory conjugate”, “nucleic acid molecules encoding the KLK5/KLK7inhibitory conjugate”, or “nucleic acid molecules encoding theKLK5/KLK14 inhibitory conjugate”, respectively), a recombinant vectorwith such a gene inserted, a cell with the gene or the vector introduced(which will be hereinafter referred to as a “cell containing nucleicacid molecules encoding the KLK5 inhibitory conjugate”, a “cellcontaining nucleic acid molecules encoding the KLK5/KLK7 inhibitoryconjugate”, or a “cell containing nucleic acid molecules encoding theKLK5/KLK14 inhibitory conjugate”), and a cell producing the KLK5inhibitory peptide conjugate, the KLK5/KLK7 inhibitory peptideconjugate, or the KLK5/KLK14 inhibitory peptide conjugate (which will behereinafter referred to as a “cell producing the KLK5 inhibitoryconjugate”, a “cell producing the KLK5/KLK7 inhibitory conjugate”, or a“cell producing the KLK5/KLK14 inhibitory conjugate”, respectively).

Suitable examples of the nucleic acid molecules encoding the KLK5inhibitory conjugate, the nucleic acid molecules encoding the KLK5/KLK7inhibitory conjugate, or the nucleic acid molecules encoding theKLK5/KLK14 inhibitory conjugate of the present invention canrespectively include those containing the nucleotide sequence describedin any one of (a1) to (a4), (b1) to (b4), or (c1) to (c4) below (whichwill be hereinafter referred to as “the nucleotide sequence of the KLK5inhibitory conjugate”, “the nucleotide sequence of the KLK5/KLK7inhibitory conjugate”, or “the nucleotide sequence of the KLK5/KLK14inhibitory conjugate”, respectively), those composed of a nucleotidesequence containing the nucleotide sequence of the KLK5 inhibitoryconjugate, the nucleotide sequence of the KLK5/KLK7 inhibitoryconjugate, or the nucleotide sequence of the KLK5/KLK14 inhibitoryconjugate, or those composed of the nucleotide sequence of the KLK5inhibitory conjugate, the nucleotide sequence of the KLK5/KLK7inhibitory conjugate, or the nucleotide sequence of the KLK5/KLK14inhibitory peptide:

(a1) a nucleotide sequence encoding the amino acid sequence set forth inany one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, and 96 (FIGS. 42,44, 46, 48, 50, 52, 54, 56, and 106) or the nucleotide sequencedescribed in any one of SEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, and95 (FIGS. 41, 43, 45, 47, 49, 51, 53, 55, and 105);(a2) a nucleotide sequence that hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence described in (a1) understringent conditions and encodes the amino acid sequence contained in apeptide or a conjugate having a KLK5 inhibitory activity;(a3) a nucleotide sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue in thenucleotide sequence described in (a1) and encodes the amino acidsequence contained in a peptide or a conjugate having a KLK5 inhibitoryactivity; and(a4) a nucleotide sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the nucleotide sequencedescribed in (a1) and encodes the amino acid sequence contained in apeptide or a conjugate having a KLK5 inhibitory activity:(b1) a nucleotide sequence encoding the amino acid sequence set forth inany one of SEQ ID NOs: 50, 52, 54, and 56 (FIGS. 58, 60, 62, and 64) orthe nucleotide sequence described in any one of SEQ ID NOs: 49, 51, 53,and 55 (FIGS. 57, 59, 61, and 63);(b2) a nucleotide sequence that hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence described in (b1) understringent conditions and encodes the amino acid sequence contained in apeptide or a conjugate having a KLK5/KLK7 inhibitory activity;(b3) a nucleotide sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue in thenucleotide sequence described in (b1) and encodes the amino acidsequence contained in a peptide or a conjugate having a KLK5/KLK7inhibitory activity; and(b4) a nucleotide sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the nucleotide sequencedescribed in (b1) and encodes the amino acid sequence contained in apeptide or a conjugate having a KLK5/KLK7 inhibitory activity:(c1) a nucleotide sequence encoding the amino acid sequence set forth inany one of SEQ ID NOs: 58 and 60 (FIGS. 66 and 68) or the nucleotidesequence described in SEQ ID NO: 57 or 59 (FIG. 65 or 67);(c2) a nucleotide sequence that hybridizes with a nucleotide sequencecomplementary to the nucleotide sequence described in (c1) understringent conditions and encodes the amino acid sequence contained in apeptide or a conjugate having a KLK5/KLK14 inhibitory activity;(c3) a nucleotide sequence that is formed by substituting, deleting,adding, and/or inserting 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue in thenucleotide sequence described in (c1) and encodes the amino acidsequence contained in a peptide or a conjugate having a KLK5/KLK14inhibitory activity; and(c4) a nucleotide sequence that is 60%, 70%, 80%, 85%, 90%, 92%, 94%,96%, 97%, 98% or 99% or more identical to the nucleotide sequencedescribed in (c1) and encodes the amino acid sequence contained in apeptide or a conjugate having a KLK5/KLK14 inhibitory activity.

A SPINK2 mutant peptide composed of an amino acid sequence encoded bythe nucleotide sequence described in any one of (a1) to (a4), (b1) to(b4), or (c1) to (c4) above or containing an amino acid sequence thatinhibits the protease activity of KLK5, KLK5 and KLK7, or KLK5 andKLK14, preferably that specifically inhibits the protease activitythereof.

However, the nucleic acid molecules encoding the KLK5 inhibitorypeptide, the KLK5/KLK7 inhibitory peptide, or the KLK5/KLK14 inhibitorypeptide are not limited to those described in (a1) to (a4), (b1) to(b4), or (c1) to (c4), and nucleic acid molecules containing anucleotide sequence encoding an amino acid sequence contained in aconjugate containing the amino acid sequence contained in a SPINK2mutant having a KLK5 inhibitory activity, KLK5/KLK7 inhibitory activity,or KLK5/KLK14 inhibitory activity, preferably, having the amino acidsequence set forth in SEQ ID NO: 61 (FIG. 69) are all included withinthe scope of the nucleic acid molecules encoding the KLK5 inhibitoryconjugate, the nucleic acid molecules encoding the KLK5/KLK7 inhibitoryconjugate, or the nucleic acid molecules encoding the KLK5/KLK14inhibitory conjugate.

For designing a nucleotide sequence encoding an amino acid sequence, oneor more codons corresponding to the respective amino acids can be used.Therefore, a base sequence encoding a single amino acid sequence of apeptide may have multiple variations. When selecting such codons, codonscan be appropriately selected corresponding to the codon usage of a hostcell for expression, into which a polynucleotide containing thenucleotide sequence or a vector containing the same is to be introduced,or the frequency or proportion of use of a plurality of codons can beappropriately adjusted. For example, in the case of using Escherichiacoli as a host cell, the nucleotide sequence may be designed usingcodons that are frequently used in Escherichia coli.

The nucleic acid molecules encoding the peptide of the present inventionor the conjugate thereof may be functionally linked to one or moreregulatory sequences. Being “functionally linked” means enabling thelinked nucleic acid molecules to be expressed or enabling the nucleotidesequence contained in the molecules to be expressed. Such a regulatorysequence contains sequence elements including information ontranscriptional regulation and/or translational regulation. Theregulatory sequence varies depending on the species but generallycontains a promoter and a 5′ non-coding sequence involved in theinitiation of transcription and translation such as a prokaryotic−35/−10 box, Shine-Dalgarno sequence, a eukaryotic TATA box, CAATsequence, and 5′ capping sequence. Such a sequence may include anenhancer element and/or a repressor element, and a translatable signalsequence, a leader sequence, and the like for delivering a natural ormature peptide to a specific compartment inside or outside the hostcell. Further, such a regulatory sequence may include a 3′ non-codingsequence, and the sequence can include elements involved intranscription termination, polyadenylation, or the like. However, when asequence relating to transcription termination does not sufficientlyfunction in a specific host cell, the sequence can be substituted with asequence suitable for the cell.

Examples of the promoter sequence can include a tet promoter, a lacUV5promoter, and a T7 promoter for prokaryotic cells, and an SV40 promoterand a CMV promoter for eukaryotic cells.

The nucleic acid molecules encoding the peptide of the present inventionor the conjugate thereof may be isolated or contained in a vector orother cloning vehicles (which will be hereinafter referred to simply asa “vector” such as a plasmid, phagemid, phage, baculovirus, and cosmid),or a chromosome, but there is no limitation to such forms. The vectormay contain a replication sequence and a control sequence that aresuitable for the host cell to be used for expression, and a selectablemarker that gives a phenotype capable of selecting a cell into which thenucleic acid molecules have been introduced by transformation or thelike, in addition to the regulatory sequence.

The nucleic acid molecules encoding the peptide of the present inventionor the conjugate thereof, and the vector containing the nucleotidesequence of the peptide of the present invention or the conjugatethereof can be introduced by methods known to those skilled in the art,such as transformation into a host cell capable of expressing thepeptide, the conjugate, or the nucleotide sequence. The host cell withthe nucleic acid molecules or the vector introduced therein can becultured under conditions suitable for expression of the peptide or thenucleotide sequence. The host cell may be either prokaryotic oreukaryotic. Examples of the prokaryotic cell can include Escherichiacoli and Bacillus subtilis, and examples of the eukaryotic cell caninclude yeasts such as Saccharomyces cerevisiae and Pichia pastoris,insect cells such as SF9 and High5, and animal cells such as HeLa cells,CHO cells, COS cells, and NS0. The peptide of the present inventionwhich is expressed can be subjected to desired post-translationalmodification using a eukaryotic cell or the like as the host cell.Examples of the post-translational modification can include the additionof functional groups such as sugar chains, the addition of peptides orproteins, the conversion of chemical properties of amino acids, and thelike. Further, desired modifications can be artificially applied to thepeptide of the present invention or the conjugate thereof. Such modifiedpeptides or conjugates are also included within the scope of the“peptide” or “conjugate” of the present invention.

The present invention also provides a method for producing a peptide ora conjugate. The method includes step 1 of culturing a cell containingnucleic acid molecules encoding a KLK5 inhibitory peptide (or a KLK5inhibitory conjugate) or a cell producing a KLK5 inhibitory peptide (ora KLK5 inhibitory conjugate), a cell containing nucleic acid moleculesencoding a KLK5/KLK7 inhibitory peptide (or a KLK5/KLK7 inhibitoryconjugate) or a cell producing a KLK5/KLK7 inhibitory peptide (or aKLK5/KLK7 inhibitory conjugate), or a cell containing nucleic acidmolecules encoding a KLK5/KLK14 inhibitory peptide (or a KLK5/KLK14inhibitory conjugate) or a cell producing a KLK5/KLK14 inhibitorypeptide (or a KLK5/KLK14 inhibitory conjugate), and/or step 2 ofcollecting a SPINK2 mutant from the culture obtained in step 1.Operations known to those skilled in the art such as fractionation,chromatography, and purification can be applied to step 2. For example,purification by affinity chromatography using an antibody of the presentinvention or a binding fragment thereof, which will be described below,can be applied thereto.

In some aspects of the invention, the peptide or a peptide included inthe conjugate has intramolecular disulfide bonds. It may be preferableto deliver the peptide having intramolecular disulfide bonds to a cellcompartment having an oxidative redox environment using a signalsequence or the like. The oxidative environment can be provided by theperiplasm of gram-negative bacteria such as Escherichia coli, theextracellular environment of gram-positive bacteria, the lumen of theendoplasmic reticulum of eukaryotic cells, or the like, and formation ofstructural disulfide bonds can be promoted under such an environment.Further, it is also possible to produce a peptide having intramoleculardisulfide bonds in the cytoplasm of a host cell such as Escherichiacoli. In such a case, the peptide can be directly acquired in a solubleand folded state or can be collected in the form of an inclusion bodyand then reconstituted in vitro. Further, it is also possible to selecta host cell having an oxidative intracellular environment to produce apeptide having intramolecular disulfide bonds in the cytoplasm thereof.Meanwhile, when the peptide has no intramolecular disulfide bond, thepeptide can be produced in a cell compartment having a reducing redoxenvironment, such as the cytoplasm of gram-negative bacteria.

The peptide of the present invention or the conjugate thereof (thepeptide moiety contained therein) can be produced by other methods knownto those skilled in the art, such as the solid-phase peptide synthesismethod of Merrifield, et al., and chemical synthesis exemplified by anorganic synthetic chemical peptide synthesis method usingt-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), or the like,and in-vitro translation.

In some aspects, the present invention provides an antibody binding tothe peptide of the present invention or a peptide contained in theconjugate, and a binding fragment thereof. The antibody may be either apolyclonal antibody or a monoclonal antibody, and the monoclonalantibody is not specifically limited, as long as it is an immunoglobulinor a derivative thereof. The binding fragment of the antibody is notlimited, as long as it has an antigen-binding activity, that is, abinding activity to the peptide. Both or one of the heavy chains andlight chains or fragments thereof, those lacking a constant region or Fcregion, and conjugates with other proteins or labeling substances arealso included therein. Such an antibody and a binding fragment thereofcan be prepared by methods known to those skilled in the art and areuseful for purification of the peptide by affinity chromatography,detection of the peptide in clinical tests, diagnoses, or the likerelated to a pharmaceutical composition containing the peptide or usethereof, immunological assay, and the like. The antibody of the presentinvention or the binding fragment thereof can be purified by affinitychromatography using the peptide of the present invention to which theantibody or the fragment binds.

5. Pharmaceutical Composition

The present invention also provides a pharmaceutical compositioncontaining the peptide of the present invention or the conjugatethereof.

The pharmaceutical composition containing the peptide of the presentinvention or the conjugate thereof is useful for treating and/orpreventing various diseases which are induced or exacerbated by KLK5(which will be hereinafter referred to as “diseases related to KLK5” or“KLK5-related diseases”) and in which suppression of such induction orexacerbation, recovery, maintenance or amelioration of symptoms,avoidance of secondary diseases, or the like is possible by inhibitingor suppressing the expression or functions of KLK5. Examples of thediseases related to KLK5 can include Netherton syndrome (Furio, L., etal. (2015) PLoS. Genet., Vol. 11, p. e1005389), atopic dermatitis(Fortugno, P., et al. (2012) Hum. Mol. Genet., Vol. 21, pp. 4187-4200),rosacea (Yamasaki, K., et al. (2007) Nat. Med., Vol. 13, pp. 975-980),UV-induced skin injury (Nin, M., et al. (2009) J. Dermatol. Sci., Vol.54, pp. 17-24), psoriasis (Komatsu, N., et al. (2007) Br. J. Dermatol.,Vol. 156, pp. 875-883), asthma (Grunberg, M., et al. (2018) Eur. J.Immunol., Vol. 48, pp. 1592-1594), spinal cord injury (Radulovic, M., etal. (2013) J. Neuropathol. Exp. Neurol., Vol. 72, pp. 1072-1089), cancer(such as uterine cancer, bladder urothelial cancer, colorectal cancer,oral squamous cell carcinoma, breast cancer, head and neck cancer,melanoma, prostate cancer, and glioma) (Emami, N., et al. (2007) Mol.Oncol., Vol. 1, pp. 269-287), and Barrett's esophagus (Gene ExpressionOmnibus, based on accession #GSE13083), but there is no limitation tothese examples.

KLK5 is considered to be the main factor in the development of Nethertonsyndrome-like skin symptoms. KLK5 is an autoactivated protease and isalso involved in activation of KLK7 and KLK14. Meanwhile, in the stratumcorneum of Netherton syndrome patients and Netherton syndrome modelmice, high protease activities like trypsin and chymotrypsin areobserved, and it is suggested that the kallikrein family locateddownstream, such as KLK7 and KLK14, are related to the protease activityin the stratum corneum, in addition to KLK5. It is expected that theremay be cases where the Netherton syndrome-like skin symptoms can be morestrongly suppressed by inhibiting KLK7 or KLK14 in addition to KLK5. 70or more examples of mutations in SPINK5 associated with Nethertonsyndrome are listed in Human Gene Mutation Database (HGMD) and reportedto be related to the severity of Netherton syndrome. Mutations in exons1 to 9 of SPINK5 are related to more severe pathologies of Nethertonsyndrome. Whether or not the pharmaceutical composition containing thepeptide of the present invention or the conjugate thereof should be usedfor treating or preventing Netherton syndrome can be determined byinvestigating the mutations in SPINK5.

The pharmaceutical composition of the present invention can contain atherapeutically or prophylactically effective amount of the peptide orthe conjugate and pharmaceutically acceptable diluents, carriers,solubilizers, emulsifiers, preservatives, and/or auxiliary agents.

The term “therapeutically or prophylactically effective amount” means anamount that exerts a therapeutic or prophylactic effect for a specificdisease, administration form, or administration route, and has the samemeaning as a “pharmacologically effective amount”.

The pharmaceutical composition of the present invention can containmaterials for changing, maintaining, or retaining the pH, the osmoticpressure, the viscosity, the transparency, the color, the isotonicity,the sterility, or the stability, the solubility, the sustained release,the absorbability, the permeability, the dosage form, the strength, theproperties, the shape, etc., of the composition or the peptide, theconjugate, or the like contained in the composition (which will behereinafter referred to as “pharmaceutical materials”). Thepharmaceutical materials are not specifically limited, as long as theyare pharmacologically acceptable materials. For example, no or lowtoxicity is a property preferably possessed by the pharmaceuticalmaterials.

Examples of the pharmaceutical materials can include, but are notlimited to, the following: amino acids such as glycine, alanine,glutamine, asparagine, histidine, arginine, or lysine, antibacterialagents, antioxidants such as ascorbic acid, sodium sulfate, or sodiumbisulfite, buffers such as phosphate, citrate, or borate buffers, sodiumbicarbonate, or tris-hydrochloric acid (Tris-HCl) solution, fillers suchas mannitol and glycine, chelating agents such asethylenediaminetetraacetic acid (EDTA), complexing agents such ascaffeine, polyvinyl pyrrolidine, β-cyclodextrin, orhydroxypropyl-β-cyclodextrin, bulking agents such as glucose, mannose,or dextrin, monosaccharides, disaccharides, other carbohydrates such asglucose, mannose, or dextrin, colorants, flavoring agents, diluents,emulsifiers, hydrophilic polymers such as polyvinyl pyrrolidine,preservatives such as low-molecular weight polypeptides, salt-formingcounterions, benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid, or hydrogen peroxide, solvents such asglycerin, propylene glycol, or polyethylene glycol (PEG), sugar alcoholssuch as mannitol or sorbitol, suspending agents, polysorbates such assorbitan ester, polysorbate 20, or polysorbate 80, surfactants such astriton, tromethamine, lecithin, or cholesterol, stability enhancers suchas sucrose or sorbitol, elasticity enhancers such as sodium chloride,potassium chloride, mannitol, or sorbitol, transport agents, diluents,excipients, and/or pharmaceutical auxiliary agents.

The amount of these pharmaceutical materials to be added is 0.001 to1000 times, preferably 0.01 to 100 times, more preferably 0.1 to 10times, the weight of the peptide of the present invention or the peptidecontained in the conjugate.

A liposome containing the peptide of the present invention or theconjugate and a pharmaceutical composition containing a modified formformed by binding the peptide to the liposome are also included withinthe pharmaceutical composition of the present invention.

The excipients or carriers are not particularly limited as long as theyare liquid or solid materials usually used for oral or parenteraladministration, such as injectable water, saline, artificialcerebrospinal fluids, and other preparations. Examples of saline caninclude neutral saline and serum albumin-containing saline.

Examples of the buffers can include a Tris buffer adjusted to bring thefinal pH of the pharmaceutical composition to 7.0 to 8.5, an acetatebuffer adjusted to bring the final pH thereof to 4.0 to 5.5, a citratebuffer adjusted to bring the final pH thereof to 5.0 to 8.0, and ahistidine buffer adjusted to bring the final pH thereof to 5.0 to 8.0.

The pharmaceutical composition of the present invention is a solid, aliquid, a suspension, or the like. Another example of the pharmaceuticalcomposition of the present invention can include a freeze-driedpreparation. The freeze-dried preparation can be formed using anexcipient such as sucrose.

The administration route of the pharmaceutical composition of thepresent invention may be any of eye drops, enteral administration,topical administration, and parenteral administration. Examples thereofcan include eye drops on the conjunctiva, intravitreal administration,intravenous administration, intraarterial administration, intramuscularadministration, intradermal administration, subcutaneous administration,intraperitoneal administration, transdermal administration, intraosseousadministration, and intraarticular administration.

The composition of the pharmaceutical composition can be determineddepending on the administration method, the inhibitory activity of thepeptide of the present invention or the peptide contained in theconjugate to KLK5, KLK5 and KLK7, or KLK5 and KLK14, the bindingaffinity, or the like. The stronger the inhibitory activity (the smallerthe IC₅₀ value or K_(i) value) or the higher the affinity (the smallerthe K_(D) value) to the target of the inhibitory peptide of the presentinvention, then the efficacy can be exerted with a lower dose.

The dose of the peptide of the present invention or the conjugatethereof is not limited, as long as it is a pharmacologically effectiveamount, and can be appropriately determined depending on the species ofthe individual, the type of disease, the symptom, the gender, the age,the chronic disease, the inhibitory activity against the target of thepeptide, the binding affinity, and other factors, but the peptide of thepresent invention or the conjugate thereof is generally administered at0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, once, twice, or moreper day for 1 to 180 days.

Examples of the form of the pharmaceutical composition can includeinjections (including freeze-dried preparations and drops),suppositories, transnasal absorption preparations, transdermalabsorption preparations, sublingual formulations, capsules, tablets,ointments, granules, aerosols, pills, powders, suspensions, emulsions,eye drops, and biological implant formulations.

The pharmaceutical composition containing the peptide of the presentinvention or the conjugate thereof as an active ingredient can beadministered simultaneously or separately with other drugs. For example,the pharmaceutical composition containing the peptide of the presentinvention or the conjugate thereof as an active ingredient may beadministered after administration of other drugs, other drugs may beadministered after administration of the pharmaceutical composition, orthe pharmaceutical composition may be administered simultaneously withother drugs. In the case of simultaneous administration, the peptide ofthe present invention or the conjugate thereof and other drugs may becontained in either a single preparation or separate preparations (aplurality of preparations).

One, two, three, or more of such other drugs can be administered orreceived. These are collectively referred to as “combined use of otherdrugs” with or “combination with other drugs” of the pharmaceuticalcomposition of the present invention. The pharmaceutical composition ofthe present invention containing other drugs in addition to the peptideof the present invention or the conjugate thereof or used in combinationwith other therapies is also included within the present invention as anaspect of “combined use of other drugs” or “combination with otherdrugs”.

Examples thereof against Netherton syndrome can include humectants,steroid drugs, and antibacterial agents. Examples thereof against atopicdermatitis can include steroid drugs, calcineurin inhibitors, PDE4inhibitors, immunosuppressive drugs, IL-4/IL-13 inhibitors, andphototherapy. Examples thereof against rosacea can include doxycycline,minocycline, azelaic acid, and brimonidine. Examples thereof againstpsoriasis can include MFG (inhibitors, IL-12/23 inhibitors, IL-17inhibitors, PDE4 inhibitors, antimetabolites, calcineurin inhibitors,fumarate ester, retinoid preparations, steroid drugs, vitamin D3analogs, and phototherapy. Examples thereof against asthma can includesteroid drugs and 02 agonists. Examples thereof against cancers such asuterine cancer, bladder urothelial cancer, colorectal cancer, oralsquamous cell carcinoma, breast cancer, head and neck cancer, melanoma,prostate cancer, and glioma can include various anticancer agents.

The present invention also provides a method for treating or preventingdiseases related to KLK5, the method comprising a step of administeringthe peptide of the present invention or the conjugate thereof, use ofthe peptide of the present invention or the conjugate thereof forpreparing a pharmaceutical composition for treating or preventing thediseases, and use of the peptide or the conjugate for treating andpreventing the diseases. A treatment or prevention kit containing thepeptide or the conjugate is also included within the present invention.

Further, a pharmaceutical composition containing a polynucleotidecontaining the nucleotide sequence encoding the amino acid sequence ofthe peptide of the present invention or the conjugate thereof, a vectorcontaining the polynucleotide, a cell containing the polynucleotide orthe vector, or a cell expressing the peptide of the present invention orthe conjugate thereof is also provided. For example, the polynucleotideand the vector can be applied to gene therapy of the diseases related toKLK5, and the cells can be applied to cell therapy of the diseasesrelated to KLK5, respectively, using known methods. Further, the cellsfor cell therapy can be prepared, for example, by introducing thepolynucleotide or the vector into autologous cells or allogeneic cells(the same kind of cells). The polynucleotide and the vector are includedwithin the present invention also as compositions for preparing celltherapeutic agents. However, the aspect of the pharmaceuticalcomposition containing the polynucleotide, the vector, the cell, or thelike of the present invention is not limited to the above.

An animal model can be used as a means for evaluating the therapeuticeffect, against diseases related to KLK5, of the peptide or theconjugate thereof contained in the pharmaceutical composition of thepresent invention as an active ingredient. Examples of a Nethertonsyndrome model having a mutation in the SPINKS gene that is thecausative gene of Netherton syndrome can include SPINKS gene-deficientmice (Descargues, P., et al. (2004) Nat. Genet., Vol. 37, pp. 56-65),SPINKS gene conditional knockout mice (Petrova, E., et al. (2019) oralpresentation in the 8th International Symposium on Kallikreins andKallikrein-Related Peptidases: Abstract Book, p. 28), and Crusty2 mice(Mutagenetix database), but there is no limitation to these examples.

6. Diagnostic Composition

A composition for testing or diagnosis containing the peptide of thepresent invention or the conjugate thereof (which will be hereinafterreferred to collectively as the “diagnostic composition”) is provided.

The diagnostic composition of the present invention is useful fortesting or diagnosing diseases related to KLK5, KLK5 expression, KLK7expression, KLK14 expression, or the like. In the present invention,examples of the test or diagnosis include determination or measurementof morbidity risk, determination of morbidity, measurement of the degreeof progression or exacerbation, measurement or determination of theeffect of drug treatment with the pharmaceutical composition containingthe peptide of the present invention or the conjugate thereof,measurement or determination of the effect of treatments other than thedrug treatment, measurement of recurrence risk, and determination ofrecurrence. However, there is no limitation to these examples, as longas they are tests or diagnoses.

The diagnostic composition of the present invention is useful foridentifying an individual to whom the peptide of the present inventionor the conjugate thereof, the composition containing the same, or thepharmaceutical composition containing the same is to be administered.

Such a diagnostic composition can contain pH buffers, osmoregulators,salts, stabilizers, preservatives, developers, sensitizers, aggregationinhibitors, and the like.

The present invention also provides a method for testing or diagnosingdiseases related to KLK5, use of the peptide of the present inventionfor preparing a diagnostic composition for the diseases, and use of thepeptide of the present invention for testing or diagnosing the diseases.A kit for testing or diagnosis containing the peptide of the presentinvention is also included within the present invention.

As regards a method for testing or diagnosis using the peptide of thepresent invention, sandwich ELISA is desirable, but detection methodssuch as conventional ELISA or RIA, ELISPOT (Enzyme-Linked ImmunoSpot),dot blotting, the Ouchterlony method, CIE(Counterimmunoelectrophoresis), CLIA (Chemiluminescent immuno assay),and FCM (Flow Cytometry) can be used. For detection, antibodies orbinding fragments thereof, or those labeled with the peptide of thepresent invention or the conjugate thereof are used. For labeling,labeling methods that can be used for biochemical analysis such aslabeling with fluorophores such as HRP, alkaline phosphatase, and FITC,radioisotopes, or the like can be used, in addition to biotin. Fordetection using an enzyme label, chemiluminescence substrates can beused, in addition to chromogenic substrates such as TMB (3, 3′, 5,5′-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolyl phosphate),p-NPP (p-nitrophenyl phosphate), OPD (o-Phenylenediamine), ABTS(3-Ethylbenzothiazoline-6-sulfonic acid), and SuperSignal ELISA PicoChemiluminescent Substrate (Thermo Fisher Scientific), and fluorescencesubstrates such as QuantaBlu (R) Fluorogenic Peroxidase Substrate(Thermo Fisher Scientific). Samples derived from humans or non-humananimals as well as artificially treated samples such as recombinantproteins can be subjected to this assay. Examples of test samplesderived from individual organisms can include, but are not limited to,blood, synovial fluids, ascites, lymph, cerebrospinal fluids,bronchoalveolar lavage, saliva, phlegm, tissue homogenate supernatants,and tissue sections.

A sandwich ELISA kit for testing or diagnosis containing the peptide ofthe present invention may contain coloring reagents, buffers fordilution, proteins for solid phase, proteins for detection, washingsolutions, or the like as well as protein standard solutions whichcomprise the peptide of the present invention or the conjugate thereof.As a method for measuring the amount of protein bound to an antigen, anabsorption method, a fluorescence method, a luminescence method, an RI(Radioisotope) method, or the like is suitably applied, and for themeasurement, an absorption plate reader, a fluorescence plate reader, aluminescence plate reader, an RI liquid scintillation counter, or thelike is preferably used.

Further, the tests or diagnoses can be performed by methods usingimmunoprecipitation.

Further, the present invention also provides a method for detecting ormeasuring KLK5, KLK5 and KLK7, or KLK5 and KLK14 in a test sample. Thediagnostic composition of the present invention can be used for such adetection or measurement method. KLK5, KLK5 and KLK7, or KLK5 and KLK14in the test sample can be detected by bringing the peptide of thepresent invention or the conjugate thereof into contact with the testsample (step 1) and then measuring the amount of KLK5, KLK5 and KLK7, orKLK5 and KLK14 bound to the peptide or the conjugate (step 2). Examplesof step 1 can include immobilizing an Fc region of an immunoglobulinconjugated to the peptide of the present invention on magnetic beads viaprotein G and adding the test sample thereto, and examples of step 2 caninclude separating the magnetic beads and analyzing the soluble proteinsprecipitated together with the beads by SDS-PAGE or Western blotting todetect KLK5, KLK5 and KLK7, or KLK5 and KLK14. Artificially treatedsamples such as recombinant proteins can be subjected to thismeasurement, in addition to human- or nonhuman animal-derived samples.Examples of test samples derived from individual organisms can include,but are not limited to, blood, synovial fluids, ascites, lymph,cerebrospinal fluids, bronchoalveolar lavage, saliva, phlegm, tissuehomogenate supernatants, and tissue sections.

The detection of KLK5, KLK5 and KLK7, or KLK5 and KLK14 can be performednot only in vitro but also in vivo. In the case of diagnostic imaging,the peptide of the present invention or the conjugate thereof labeledwith a pharmaceutically acceptable radionuclide or luminescent materialcan be used. Examples of step 1 can include administering the labeledpeptide or the conjugate thereof to a test subject, and examples of step2 can include capturing an image using a diagnostic imaging techniquesuch as PET/CT and determining or testing for the presence of KLK5, KLK5and/or KLK7, or KLK5 and/or KLK14.

The peptide or the conjugate thereof contained in the diagnosticcomposition of the present invention binds to KLK5, KLK5 and KLK7, orKLK5 and KLK14, and preferably has KLK5-, KLK5 and KLK7-, or KLK5 andKLK14-specific binding activity.

A method for identifying an individual to whom the pharmaceuticalcomposition of the present invention is to be administered is alsoincluded within the present invention. In such an identification method,KLK5, KLK5 and/or KLK7, or KLK5 and/or KLK14 in a sample derived fromthe individual is measured, and the individual can be determined to bepositive when KLK5, KLK5 and/or KLK7, or KLK5 and/or KLK14 is detectedin the sample, or KLK5, KLK5 and/or KLK7, or KLK5 and/or KLK14 isdetected in an amount larger than the amount of KLK5, KLK5 and/or KLK7,or KLK5 and/or KLK14 detected in another sample derived from a healthyindividual. For this method, the diagnostic composition of the presentinvention can be used.

Further, in a suitable aspect of the identification method, theindividual has or is at risk of KLK5-related diseases.

Further, in one aspect, the pharmaceutical composition of the presentinvention can be administered to an individual determined to be positivein the identification method.

7. Method for separating KLK5, KLK5 and KLK7, or KLK5 and KLK14

The peptide of the present invention or the conjugate thereof preferablyhas a binding activity specific to KLK5, KLK5 and/or KLK7, or KLK5and/or KLK14. Accordingly, KLK5, KLK5 and/or KLK7, or KLK5 and/or KLK14can be specifically separated from a sample in which KLK5, KLK5 and/orKLK7, or KLK5 and/or KLK14 is mixed with other KLKs, using the peptideof the present invention or the conjugate thereof. The release of KLK5,KLK5 and/or KLK7, or KLK5 and/or KLK14 from the peptide or the conjugatecan be performed non-selectively, for example, under a relatively highionic strength, a low pH, moderate denaturing conditions, in thepresence of chaotropic salts, or the like, but is preferably performedwithin a range in which the protease activity of KLK5, KLK5 and/or KLK7,or KLK5 and/or KLK14 is not attenuated.

EXAMPLES

In the following examples, some aspects of the present invention will bedescribed more specifically. However, the present invention is notlimited to them.

In the following examples, operations relating to genetic engineeringwere performed according to the methods described in “Molecular Cloning”(Sambrook, J., Fritsch, E. F., and Maniatis, T., Cold Spring HarborLaboratory Press, published in 1982 or published in 1989) and othermethods described in experimental manuals used by those skilled in theart, or according to the instructions of commercially available productsin the cases where commercially available reagents or kits were used,unless otherwise indicated.

Example 1. Preparation of KLK5 Inhibitory Peptide (1-1) Construction ofKLK5 Inhibitory Peptide Expression Vector

Using the nucleotide sequence of each inhibitory peptide (SEQ ID NO: 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31) and the nucleotidesequence of SPINK2 as templates, inhibitory peptide fragments wereamplified by PCR ((at 94° C. for 15 seconds, at 60° C. for 30 seconds,and at 68° C. for 20 seconds)×30 cycles) using the following primers andKOD-plus-(TOYOBO).

Primer 1: 5′-AAAAGGATCCCTGGACAAACGTGGCCCGCAGTTTGGTCTGTTTAG-3′ (SEQ IDNO: 62, FIG. 70) Primer 2: 5′-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAA-3′(SEQ ID NO: 63, FIG. 71)

The amplified fragments were subjected to agarose gel electrophoresis,and then the desired DNA fragments were cut out, to prepare DNA using aQIAquick Gel Extraction Kit (QIAGEN). The prepared DNA fragments and pET32a (Novagen) were treated using restriction enzymes BamHI (NEB) andXhoI (NEB) at 37° C. for 1 hour or more, and after agarose gelelectrophoresis, the desired DNA fragments were cut out, followed bypurification using a QIAquick PCR Purification Kit (QIAGEN). Using aLigaFast Rapid DNA Ligation System (Promega), each purified fragment wasreacted at room temperature for 10 minutes to conduct a ligationreaction. The ligation solution was added to Escherichia coli JM109(TOYOBO), left standing on ice for 30 minutes, then heat-treated at 42°C. for 45 seconds, further left standing on ice for 5 minutes, seeded ona 2YT plate containing 0.1 mg/mL ampicillin, and thereafter staticallycultured at 37° C. overnight, to transform the Escherichia coli. Thenext day, the transformed Escherichia coli was inoculated on a TerrificBroth medium (Invitrogen) containing 0.1 mg/mL ampicillin and culturedat 37° C. overnight. Thereafter, plasmid DNA was collected using aQIAprep 96 Turbo Miniprep Kit (Qiagen) (which will be hereinafterreferred to as “miniprep treatment”), and a sequence analysis wasconducted, to construct a pET 32a_Kex2_KLK5 inhibitory peptide vector.

(1-2) Preparation of KLK5 Inhibitory Peptide

Escherichia coli Origami B (DE3) (Novagen) was transformed with thevector constructed in (1-1) and cultured at 37° C. using a 2YT mediumcontaining 0.1 mg/mL ampicillin. Thereafter, IPTG (with a finalconcentration of 1 mM) was added thereto, followed by culture at 16° C.overnight. The next day, after collecting the cells by centrifugation(3,000 g, 20 minutes, and 4° C.), a lysate was prepared using BugBusterMaster Mix (Novagen), and a His tag fusion target protein was purifiedusing TALON Metal Affinity Resin (Clontech). Thereafter, a thioredoxintag was cleaved from the desired protein using Kex2 (Saccharomycescerevisiae: Accession CAA96143) and purified using TALON. Further, gelfiltration chromatography (Superdex75 10/300 GL) or reverse phasechromatography (YMC-Pack ODS-AM) was applied, to prepare 14 kinds ofKLK5 inhibitory peptides. The amino acid sequences of the derivativesare shown in SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, and 32 (FIGS. 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,and 40).

Example 2. Preparation of KLK5, KLK7, and KLK14 (2-1) Construction ofHuman KLK5, Human KLK7, and Human KLK14 Expression Vectors

The primers and PCR conditions used for cloning human pro-KLK5, humanpro-KLK7, and human pro-KLK14 were as follows. Fragment A was amplifiedby PCR ((at 94° C. for 15 seconds, at 60° C. for 30 seconds, and at 68°C. for 10 seconds)×30 cycles) using the following primers andKOD-plus-(TOYOBO).

Primer 3: 5′-GGCGATTATAAAGATGACGATGATAAACACCATCACCACCATC-3′(SEQ ID NO: 64, FIG. 72)  Primer 4:5′-GTTTAAACTCAATGATGGTGGTGATGGTGTTTATCATCGTCAT-3′(SEQ ID NO: 65, FIG. 73) 

Next, using nucleotide sequences encoding human pro-KLK5 (Uniprot:Q9Y337), human pro-KLK7 (Uniprot: P49862), and human pro-KLK14 (Uniprot:Q9P0G3) respectively as templates, fragments were amplified by PCR ((at94° C. for 15 seconds, at 60° C. for 30 seconds, and at 68° C. for 60seconds)×30 cycles) using the following primers and KOD-plus-(TOYOBO).

Human pro-KLK5 amplification primer  Primer 5:5′-AAAATCTAGAGCCGCCACCATGGCCACAGCTAGACCCCCT-3′ (SEQ ID NO: 66, FIG. 74) Primer 6: 5′-CGTCATCTTTATAATCGCCGCTGTTGGCCTGGATGGTTTCCTG-3′(SEQ ID NO: 67, FIG. 75)  Human pro-KLK7 amplification primer  Primer 7:5′-AAAATCTAGAGCCGCCACCATGGCCAGATCTCTGCTGCTGCCC-3′(SEQ ID NO: 68, FIG. 76)  Primer 8:5′-CGTCATCTTTATAATCGCCCCGGTGTTTCTTCATGGTGTCGTT-3′(SEQ ID NO: 69, FIG. 77)  Human pro-KLK14 amplification primer Primer 9: 5′-AAAATCTAGAGCCGCCACCATGTTCCTCCTCCTCACCGCCCTC-3′(SEQ ID NO: 70, FIG. 78)  Primer 10:5′-CGTCATCTTTATAATCGCCCTTGTCGCGCATGGTCTCCTCGAT-3′(SEQ ID NO: 71, FIG. 79) 

Desired DNA fragments were amplified by overlapping PCR using thefragments amplified above and fragment A, the following primers, andKOD-plus-(TOYOBO).

Primer 5 (SEQ ID NO: 66, FIG. 74) or primer 7 (SEQ ID NO: 68, FIG. 76)or primer 9 (SEQ ID NO: 70, FIG. 78) and

Primer 11: 5′-AAAAGTTTAAACTCAATGATGGTGGTGATGGTGT-3′(SEQ ID NO: 72, FIG. 80) 

Next, using nucleotide sequences encoding mouse pro-KLK7 (Uniprot:Q91VE3) or mouse pro-KLK14 (Uniprot: Q8CGR5) respectively as templates,fragments were amplified by PCR ((at 94° C. for 15 seconds, at 60° C.for 30 seconds, and at 68° C. for 60 seconds)×30 cycles) using thefollowing primers and KOD-plus-(TOYOBO).

Mouse pro-KLK7 amplification primer  Primer 12:5′-AAAATCTAGAGCCGCCACCATGGGAGTGTGGCTGCTGAGCCTG-3′(SEQ ID NO: 73, FIG. 81)  Primer 13:5′-AAAAGTTTAAACTCAATGATGGTGGTGATGGTGCCGGTGGGTCTTCATGGTTTCCATG-3′ (SEQ ID NO: 74, FIG. 82) Mouse pro-KLK14 amplification primer  Primer 14:5′-AAAATCTAGAGCCGCCACCATGTTTCTGCTGCTGATCATCCTG-3′(SEQ ID NO: 75, FIG. 83)  Primer 15:5′-AAAAGTTTAAACTCAATGATGGTGGTGATGGTGGTTGCTCTGCATGGTCCGCTGAA-3′ (SEQ ID NO: 76, FIG. 84) 

Mammalian cell expression vectors pCMA_pro-hKLK5, pCMA_pro-hKLK7,pCMA_pro-hKLK14, pCMA_pro-mKLK7, and pCMA_pro-mKLK14 with a His tagadded at the C-terminus encoded by each gene were constructed by cloningusing the desired DNA fragments thus amplified and restriction enzymesXbaI (NEB) and PmeI (NEB). The operation was performed according to themethod described in (1-1).

(2-2) Expression and Purification of Human KLK5, Human Pro-KLK7, HumanPro-KLK14, Mouse Pro-KLK7, and Mouse Pro-KLK14

Each expression vector constructed in (2-1) was transfected intoExpi293F cells (Thermo Fisher Scientific) using PEI MAX 40000(Polysciences), and the culture supernatant was collected after 3 daysof culturing the cells. The desired His tag fusion protein was collectedfrom the culture supernatant using HisTrap excel (GE healthcare), andthe buffer was replaced with PBS using Amicon Ultra NMWL 10,000 (MerckKGaA Millipore), to purify KLK5, human pro-KLK7, human pro-KLK14, mousepro-KLK7, and mouse pro-KLK14, respectively.

(2-3) Preparation of Human KLK5, Human KLK7, Human KLK14, Mouse KLK5,Mouse KLK7, and Mouse KLK14

To 200 μg/mL pro-KLK7 or 14 prepared using a KLK activation buffer (50mM Tris-HCl, 150 mM NaCl, 10 mM CaCl₂), 0.05% (w/w) Brij-35, pH 7.5),was added an equal amount of 20 μg/mL thermolysin, followed by reactionfor a certain time at 37° C. Thereafter, an equal amount of 100 mM EDTAwas mixed therein to prepare activated human KLK7, activated humanKLK14, activated mouse KLK7, and activated mouse KLK14.

Further, 200 μg/mL mouse KLK5 (R&D Systems, Inc., 7236-SE) and 2 μg/mLhuman KLK5 prepared using an activation buffer (50 mM Tris-HCl, 0.005%(w/w) Brij-35, pH 8.0) were mixed in equal amounts, followed by reactionat 37° C. for 24 hours, to prepare activated mouse KLK5.

Example 3. Evaluation of KLK5 Inhibitory Peptide (3-1) Evaluation ofHuman/Mouse KLK5, Human/Mouse KLK7, and Human/Mouse KLK14 InhibitoryActivities of KLK5 Inhibitory Peptide

The substrate peptide was dissolved in DMSO to 10 mM and diluted with anassay buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) for use. 25 μL eachof human/mouse KLK5, human/mouse KLK7, or human/mouse KLK14 and theinhibitory peptide diluted with the assay buffer were mixed, followed bya reaction at 37° C. for 20 minutes. Thereafter, 50 μL of a substratediluted with the assay buffer was added thereto, and the fluorescencesignal was measured using Enspire (PerkinElmer). The combinations of theenzyme and the substrate used were as follows. Each inhibitory peptidehad a final concentration of 0.098 to 1,000 nM, and a PROTEOSAVE (R)SS96F black plate (Sumitomo Bakelite Co., Ltd.) was used for thereaction and the measurement.

Evaluation of Human KLK5 inhibitory activity: hKLK5 with a finalconcentration of 10 nM, a substrate peptide Boc-Val-Pro-Arg-AMC (R&DSystems, Inc.) with a final concentration of 100 μM, and a fluorescencesignal with excitation at 380 nm/emission at 460 nmEvaluation of Human KLK7 inhibitory activity: hKLK7 with a finalconcentration of 1 μg/mL, a substrate peptideMca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys (Dnp)-NH₂ (SEQ ID NO: 101)(R&D Systems, Inc., FIG. 85, and the amino acid sequence shown in SEQ IDNO: 77) with a final concentration of 20 μM, and a fluorescence signalwith excitation at 320 nm/emission at 405 nmEvaluation of Human KLK14 inhibitory activity: hKLK14 with a finalconcentration of 0.2 μg/mL, a substrate peptide Boc-Val-Pro-Arg-AMC witha final concentration of 100 μM (R&D Systems, Inc.), and a fluorescencesignal with excitation at 380 nm/emission at 460 nmEvaluation of Mouse KLK5 inhibitory activity: mouse KLK5 with a finalconcentration of 0.25 μg/mL, a substrate peptide Boc-Val-Pro-Arg-AMCwith a final concentration of 100 μM (R&D Systems, Inc.), and afluorescence signal with excitation at 380 nm/emission at 460 nmEvaluation of Mouse KLK7 inhibitory activity: mouse KLK7 with a finalconcentration of 0.5 μg/mL, a substrate peptideMca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys (Dnp)-NH₂ (SEQ ID NO: 101)with a final concentration of 7 μM (R&D Systems, Inc., FIG. 85, and theamino acid sequence shown in SEQ ID NO: 77), and a fluorescence signalwith excitation at 320 nm/emission at 405 nmEvaluation of Mouse KLK14 inhibitory activity: mouse KLK14 with a finalconcentration of 0.1 μg/mL, a substrate peptide Boc-Val-Pro-Arg-AMC witha final concentration of 100 μM (R&D Systems, Inc.), and a fluorescencesignal with excitation at 380 nm/emission at 460 nm

As a result of calculating the degradation rate of the substrate peptideby each inhibitory peptide at each concentration and calculating the 50%inhibitory concentration (IC₅₀) using GraphPad Prism (version 5.0;GraphPad Software Inc.) with the degradation rate of the inhibitorypeptide at a concentration of 0 nM taken as 100%, it was revealed thatall of the inhibitory peptides inhibited human KLK5 enzymatic activityat low concentrations (Table 1, FIG. 2). Some of the inhibitory peptidesinhibited human KLK7 or human KLK14 enzymatic activity at lowconcentrations, and some of the inhibitory peptides exhibited weakinhibitory activities against these proteases (Table 1). The inhibitorypeptides also exhibited a similar activity against mouse KLK5, KLK7, orKLK14 (Table 2). The average value of three independent experiments wasused to calculate the IC₅₀ value.

TABLE 1 Human KLK5, human KLK7, or human KLK14 inhibitory activity ofeach KLK5 inhibitory peptide IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) ID for hKLK5for hKLK7 for hKLK14 K51028 6.1 ± 0.5 249 ± 25   >1,000 K50032 7.1 ±0.4 >1,000 >1,000 K50055 6.7 ± 0.2 >1,000 >1,000 K51069 6.8 ± 0.4 >1,00072 ± 23 K51072 7.0 ± 0.2 >1,000 434 ± 18  K50015 6.8 ± 0.3 >1,000 17 ±1  K50016 6.9 ± 1.1 >1,000 >1,000 K51034 6.4 ± 0.3 >1,000 >1,000 K500627.5 ± 0.7 >1,000 499 ± 27  K51090 4.5 ± 0.3 >1,000 >1,000 K50098 8.3 ±0.9 >1,000 268 ± 31  K51005 5.8 ± 2.1 17 ± 0.2 >1,000 K50031 5.4 ± 0.113 ± 0.3 >1,000 K51057 12 ± 1  24 ± 0.7 >1,000

TABLE 2 Mouse KLK5, mouse KLK7, or mouse KLK14 inhibitory activity ofeach KLK5 inhibitory peptide IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) ID for mKLK5for mKLK7 for mKLK14 K51028 <0.5 77 >1,000 K50032 127 >1,000 >1,000K50055 <0.5 >1,000 >1,000 K51069 <0.5 181 40 K51072 2 512 >1,000 K50015<0.5 248 108 K50016 15 >1,000 >1,000 K51034 54 >1,000 >1,000 K5006210 >1,000 >1,000 K51090 2 658 >1,000 K50098 14 >1,000 547 K51005 48 23214 K50031 40 4 244 K51057 149 27 437

(3-2) Evaluation of Cross-Reactivity of KLK5 Inhibitory Peptide

The specificities for other proteases were evaluated using thedegradation of a substrate peptide as an index. As in the methoddescribed in (3-1), 25 μL each of a protease and a sample diluted withan assay buffer were mixed (to a final concentration of sample of 1 μM),followed by a reaction at 37° C. for 20 minutes. Thereafter, 50 μL of asubstrate diluted with an assay buffer was added thereto, and thefluorescence signal was measured using Enspire (PerkinElmer). An assaybuffer (50 mM Tris, 150 mM NaCl, pH 8.0) was used for the evaluation ofthe protease activity, and a PROTEOSAVE (R) SS96F black plate (SumitomoBakelite Co., Ltd.) was used for the reaction and the measurement. Thecombinations of the protease and the substrate used for the evaluationof the specificity were as follows.

Evaluation of Bovine trypsin inhibitory activity: trypsin (Pierce;20233) with a final concentration of 5 nM and a substrate peptideBoc-Val-Pro-Arg-AMC with a final concentration of 100 μM (R&D Systems,Inc., ES011), and a fluorescence signal with excitation at 380nm/emission at 460 nmEvaluation of Human trypsin inhibitory activity: trypsin with a finalconcentration of 1 nM (Sigma-Aldrich Co. LLC, T6424) and a substratepeptide Boc-Val-Pro-Arg-AMC with a final concentration of 100 μM (R&DSystems, Inc., ES011), and a fluorescence signal with excitation at 380nm/emission at 460 nmEvaluation of Bovine α-chymotrypsin inhibitory activity: chymotrypsinwith a final concentration of 10 nM (Worthington BiochemicalCorporation; LS001434) and a substrate peptide Suc-Leu-Leu-Val-Tyr-MCA(SEQ ID NO: 98) with a final concentration of 100 μM (PEPTIDE INSTITUTE,INC., 3120-v, FIG. 86, and the amino acid sequence shown in SEQ ID NO:78), and a fluorescence signal with excitation at 380 nm/emission at 460nmEvaluation of Human chymotrypsin inhibitory activity: chymotrypsin witha final concentration of 10 nM (Sigma-Aldrich Co. LLC, C8946), asubstrate peptide Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO: 98) with a finalconcentration of 10 μM (PEPTIDE INSTITUTE, INC., 3120-v, FIG. 86, andthe amino acid sequence shown in SEQ ID NO: 78), and a fluorescencesignal with excitation at 380 nm/emission at 460 nmEvaluation of Human tryptase inhibitory activity: tryptase with a finalconcentration of 1 nM (Sigma-Aldrich Co. LLC, T7063) and a substratepeptide Boc-Phe-Ser-Arg-MCA with a final concentration of 100 μM(PEPTIDE INSTITUTE, INC., 3107-v), and a fluorescence signal withexcitation at 380 nm/emission at 460 nmEvaluation of Human chymase inhibitory activity: chymase with a finalconcentration of 100 nM (Sigma-Aldrich Co. LLC, C8118) and a substratepeptide Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO: 98) with a finalconcentration of 100 μM (PEPTIDE INSTITUTE, INC., 3120-v, FIG. 86, andthe amino acid sequence shown in SEQ ID NO: 78), and a fluorescencesignal with excitation at 380 nm/emission at 460 nmEvaluation of Human plasmin inhibitory activity: Plasmin with a finalconcentration of 50 nM (Sigma-Aldrich Co. LLC, P1867) and a substratepeptide Boc-Val-Leu-Lys-MCA with a final concentration of 100 μM(PEPTIDE INSTITUTE, INC., 3104-v), and a fluorescence signal withexcitation at 380 nm/emission at 460 nmEvaluation of Human thrombin inhibitory activity: thrombin with a finalconcentration of 1 nM (Sigma-Aldrich Co. LLC, T6884) and a substratepeptide Boc-Val-Pro-Arg-AMC with a final concentration of 100 μM (R&DSystems, Inc., ES011), and a fluorescence signal with excitation at 380nm/emission at 460 nmHuman neutrophil elastase inhibitory activity: Neutrophil elastase witha final concentration of 0.00001 U/μL (Enzo Life Sciences) and asubstrate peptide Suc (OMe)-Ala-Ala-Pro-Val-MCA (SEQ ID NO: 99) with afinal concentration of 100 μM (PEPTIDE INSTITUTE, INC., 3153-v, FIG. 87,and the amino acid sequence shown in SEQ ID NO: 79), and a fluorescencesignal with excitation at 380 nm/emission at 460 nmEvaluation of Human matriptase inhibitory activity: matriptase with afinal concentration of 1 nM (R&D Systems, Inc., 3946-SE) and a substratepeptide Boc-Gln-Ala-Arg-AMC with a final concentration of 100 μM (R&DSystems, Inc., ES014), and a fluorescence signal with excitation at 380nm/emission at 460 nmEvaluation of Human protein C inhibitory activity: protein C with afinal concentration of 100 nM (Sigma-Aldrich Co. LLC, P2200) and asubstrate peptide Boc-Leu-Ser-Thr-Arg-MCA (SEQ ID NO: 100) with a finalconcentration of 100 μM (PEPTIDE INSTITUTE, INC., 3112-v, FIG. 88, andthe amino acid sequence shown in SEQ ID NO: 80) a fluorescence signalwith excitation at 380 nm/emission at 460 nmEvaluation of Human tPA inhibitory activity: tPA with a finalconcentration of 10 nM (Sigma-Aldrich Co. LLC, 10831) and a substratepeptide Pyr-Gly-Arg-MCA with a final concentration of 100 μM (PEPTIDEINSTITUTE, INC., 3145-v), and a fluorescence signal with excitation at380 nm/emission at 460 nmEvaluation of Human uPA inhibitory activity: uPA with a finalconcentration of 2 nM (Sigma-Aldrich Co. LLC, U0633) and a substratepeptide Pyr-Gly-Arg-MCA with a final concentration of 100 μM (PEPTIDEINSTITUTE, INC., 3145-v), and a fluorescence signal with excitation at380 nm/emission at 460 nmEvaluation of Human plasma kallikrein inhibitory activity: plasmakallikrein with a final concentration of 0.125 μg/mL (R&D Systems, Inc.,2497-SE) and a substrate peptide Z-Phe-Arg-MCA with a finalconcentration of 100 μM (PEPTIDE INSTITUTE, INC., 3095-v), and afluorescence signal with excitation at 380 nm/emission at 460 nmEvaluation of Human KLK1 inhibitory activity: KLK1 with a finalconcentration of 0.1 μg/mL (R&D Systems, Inc., 2337-SE) and a substratepeptide Pro-Phe-Arg-MCA with a final concentration of 100 μM (PEPTIDEINSTITUTE, INC., 3096-v), and a fluorescence signal with excitation at380 nm/emission at 460 nmEvaluation of Human KLK2 inhibitory activity: KLK2 with a finalconcentration of 2 μg/mL (R&D Systems, Inc., 4104-SE) and a substratepeptide Pro-Phe-Arg-MCA with a final concentration of 100 μM (PEPTIDEINSTITUTE, INC., 3096-v), and a fluorescence signal with excitation at380 nm/emission at 460 nmEvaluation of Human KLK4 inhibitory activity: KLK4 with a finalconcentration of 1 μg/mL (R&D Systems, Inc., 1719-SE) and a substratepeptide Boc-Val-Pro-Arg-AMC with a final concentration of 100 μM (R&DSystems, Inc., ES011), and a fluorescence signal with excitation at 380nm/emission at 460 nmEvaluation of Human KLK7 inhibitory activity: KLK7 with a finalconcentration of 1 μg/mL, a substrate peptideMca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys (Dnp)-NH2 (SEQ ID NO: 101)with a final concentration of 20 μM (R&D Systems, Inc., FIG. 85, and theamino acid sequence shown in SEQ ID NO: 77), and a fluorescence signalwith excitation at 320 nm/emission at 405 nmEvaluation of Human KLK8 inhibitory activity: KLK8 with a finalconcentration of 5 nM (UniProt: 060259, prepared by the inventors) and asubstrate peptide Boc-Val-Pro-Arg-AMC with a final concentration of 100μM (R&D Systems, Inc., ES011), and a fluorescence signal with excitationat 380 nm/emission at 460 nmEvaluation of Human KLK12 inhibitory activity: KLK12 with a finalconcentration of 0.1 μg/mL (R&D Systems, Inc., 3095-SE) and a substratepeptide Boc-Val-Pro-Arg-AMC with a final concentration of 100 μM (R&DSystems, Inc., ES011), and a fluorescence signal with excitation at 380nm/emission at 460 nmEvaluation of Human KLK13 inhibitory activity: KLK13 with a finalconcentration of 0.5 μg/mL (R&D Systems, Inc., 2625-SE) and a substratepeptide Boc-Val-Pro-Arg-AMC with a final concentration of 100 μM (R&DSystems, Inc., ES011), and a fluorescence signal with excitation at 380nm/emission at 460 nmEvaluation of Human KLK14 inhibitory activity: hKLK14 with a finalconcentration of 0.2 μg/mL, a substrate peptide Boc-Val-Pro-Arg-AMC witha final concentration of 100 μM (R&D Systems, Inc.), and a fluorescencesignal with excitation at 380 nm/emission at 460 nm

In the same manner as in (3-1), the cross-reactivities of each KLK5inhibitory peptide to proteases other than KLK5 were evaluated using thedegradation of a peptide substrate as an index. Some of the inhibitorypeptides exhibited weak cross-reactivity to Chymotrypsin with a finalconcentration of the inhibitory peptide of 1 μM (where the IC₅₀ valuewas less than 1 μM), but most of the inhibitory peptides exhibited noinhibitory activity to any protease other than KLKn (n=1, 2, 4, 5, 7, 8,12, or 14) (FIG. 3). Meanwhile, some of the inhibitory peptidesexhibited inhibitory activity to KLK4 or KLK12 with a finalconcentration of the inhibitory peptide of 1 μM (where the IC₅₀ valuewas less than 1 μM), but many of the inhibitory peptides exhibited noprotease inhibitory activity against KLKn except KLK5, KLK7, and KLK14,and thus it was found that the inhibitory peptides had high specificity.

(3-3) Evaluation of KLK5 Binding Activity of KLK5 Inhibitory Peptide

In order to measure the binding affinity of the KLK5 inhibitorypeptides, surface plasmon resonance analysis was conducted using BIAcoreT 200 (GE healthcare). A complementary strand of DNA of a streptavidinconjugate was captured by hybridization on Sensor Chip CAP (GEhealthcare) on which single-stranded DNA immobilized. Next, KLK5biotinylated using EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific)was captured at a flow rate of 10 μL/min to immobilize about 10 RU.Thereafter, the KLK5 inhibitory peptide, serially diluted 2-fold withHBS-EP (0.625 to 10 nM), was added thereto as an analyte at a flow rateof 30 μL/min. In the BIAcore T 200 Evaluation software (version 2.0),kon and koff were calculated by analysis using Single cycle kinetics ina simple one-to-one Langmuir binding model. The dissociation constantK_(D) was calculated as a ratio of k_(off)/k_(on). Further, multipleKLK5 inhibitory peptides were measured by regenerating Sensor Chip CAPusing a Regeneration buffer attached to a Biotin CAPture Kit (GEhealthcare) and repeatedly capturing biotinylated KLK5.

All of the 14 kinds of KLK5 inhibitory peptides measured exhibited K_(D)values below 1 nM, revealing that their binding activities were verystrong (Table 3A).

TABLE 3A KLK5 binding activity of each KLK5 inhibitory peptide IDKon(1/M · s) Koff(1/s) K_(D)(M) K51028 1.5 × 10⁶ 2.0 × 10⁻⁵ 1.4 × 10⁻¹¹K50032 2.0 × 10⁶ 2.7 × 10⁻⁴ 1.3 × 10⁻¹⁰ K50055 9.8 × 10⁵ 1.8 × 10⁻⁵ 1.9× 10⁻¹¹ K51069 9.4 × 10⁵ 1.0 × 10⁻⁶ 1.0 × 10⁻¹² K51072 8.4 × 10⁵ 2.2 ×10⁻⁵ 2.6 × 10⁻¹¹ K50015 9.4 × 10⁵ 1.0 × 10⁻⁶ 1.1 × 10⁻¹² K50016 7.0 ×10⁶ 2.9 × 10⁻⁴ 4.2 × 10⁻¹¹ K51034 5.2 × 10⁵ 3.5 × 10⁻⁵ 6.8 × 10⁻¹¹K50062 1.2 × 10⁶ 2.9 × 10⁻⁶ 2.5 × 10⁻¹² K51090 2.1 × 10⁶ 5.7 × 10⁻⁵ 2.7× 10⁻¹¹ K50098 1.0 × 10⁶ 1.8 × 10⁻⁵ 1.8 × 10⁻¹¹ K51005 3.5 × 10⁶ 1.2 ×10⁻³ 3.4 × 10⁻¹⁰ K50031 8.2 × 10⁵ 5.0 × 10⁻⁵ 6.1 × 10⁻¹¹ K51057 6.0 ×10⁶ 1.5 × 10⁻³ 2.6 × 10⁻¹⁰

(3-4) Evaluation of KLK5 Binding Activity of KLK5 Inhibitory Peptide FcFusion

In order to measure the binding affinity of a KLK5 inhibitory peptide Fcfusion prepared in (5-2), which will be described below, surface plasmonresonance analysis was conducted using BIAcore T 200 (GE healthcare).

The KLK5 inhibitory peptide Fc fusion was captured at a flow rate of 20μL/min by Sensor Chip CM5 (GE healthcare) on which an anti-human IgG(Fc) antibody was immobilized to immobilize about 30 to 50 RU.Thereafter, KLK5, serially diluted 2-fold with HBS-EP (0.625 to 10 nM),was added thereto as an analyte at a flow rate of 30 μL/min. In theBIAcore T 200 Evaluation software (version 2.0), kon and koff werecalculated by analysis using Single cycle kinetics in a simpleone-to-one Langmuir binding model. The dissociation constant K_(D) wascalculated as a ratio of k_(off)/k_(on). Further, the binding activityof KLK5 to multiple KLK5 inhibitory peptide Fc fusions was measured byregenerating Sensor Chip CM5 on which the anti-human IgG (Fc) antibodywas immobilized using a Regeneration buffer attached to a Human AntibodyCapture Kit (GE healthcare) and repeatedly capturing the KLK5 inhibitorypeptide Fc fusion.

All of the 14 kinds of KLK5 inhibitory peptide Fc fusions measuredexhibited K_(D) values below 1 nM, revealing that their bindingactivities were very strong (Table 3B).

TABLE 3B KLK5 binding activity of each KLK5 inhibitory peptide Fc fusionID Kon(1/M · s) Koff(1/s) KD(M) D3-K51028-Fc 5.39 × 10⁵ 1.81 × 10⁻⁶ 3.35× 10⁻¹² D3-K50032dN-Fc 1.08 × 10⁶ 5.69 × 10⁻⁶ 5.25 × 10⁻¹² D3-K50055-Fc5.49 × 10⁵ 2.32 × 10⁻⁶ 4.22 × 10⁻¹² D3-K51069dN-Fc 7.14 × 10⁵ 2.81 ×10⁻⁶ 3.93 × 10⁻¹² D3-K51072-Fc 2.25 × 10⁵ 1.68 × 10⁻⁶ 7.48 × 10⁻¹²D3-K50015-Fc 1.04 × 10⁶ 3.52 × 10⁻⁶ 3.38 × 10⁻¹² D3-K50016dN-Fc 7.18 ×10⁵ 5.60 × 10⁻⁶ 7.81 × 10⁻¹² D3-K51034-Fc 2.83 × 10⁵ 2.93 × 10⁻⁶ 1.03 ×10⁻¹¹ D3-K50062-Fc 5.66 × 10⁵ 6.52 × 10⁻⁶ 1.15 × 10⁻¹¹ D3-K51090-Fc 7.00× 10⁵ 7.86 × 10⁻⁶ 1.12 × 10⁻¹¹ D3-K50098dN-Fc 9.22 × 10⁵ <1.62 × 10⁻⁶ <1.76 × 10⁻¹²  D3-K51005-Fc 1.47 × 10⁵ 1.96 × 10⁻⁵ 1.33 × 10⁻¹⁰D3-K50031-Fc 8.81 × 10⁴ <1.00 × 10⁻⁶  <1.14 × 10⁻¹¹  D3-K51057-Fc 8.69 ×10⁴ 1.79 × 10⁻⁵ 2.06 × 10⁻¹⁰

Example 4. Analysis of KLK5 Inhibitory Peptide Using X-Ray CrystalStructure (4-1) Preparation of KLK5/KLK5 Inhibitory Peptide Complex

According to the methods described in (1-2) and (2-2), KLK5 inhibitorypeptides K51034 and KLK5 having the amino acid sequences shown by theSEQ ID numbers were prepared. After mixing the two under conditions of50 mM Tris-HCl, 150 mM NaCl, and pH 8.0, a complex was isolated andpurified by gel filtration chromatography (Superdex 200 10/300 GL).

(4-2) Analysis of X-Ray Crystal Structure

The complex solution prepared in (4-1) was concentrated to 12 mg/mL,thereafter mixed with a reservoir solution (0.2 M Magnesium Chloridehexahydrate, 20% PEG3350) at a ratio of 1:1, and crystallized by a vapordiffusion method. The obtained cubic single crystal was immersed in areservoir solution containing 20% glycerol and thereafter frozen inliquid nitrogen. The frozen crystals were irradiated with X-rays in acryo-stream to obtain a diffraction image (Hypixel 6000HE/MicroMax007).Scaling data with a maximum resolution of 1.7 Å was obtained by analysisusing CrysAlisPro. The phase was determined by the molecular replacementmethod using KLK5 alone (PDB ID: 2PSX) and SPINK2 alone (PDB ID: 2JXD)as templates. After structural refinement, complex crystals of KLK5/thepeptide K51034 were determined at a resolution of 1.8 Å. One moleculeeach of KLK5 and SPINK2 was contained in a unit cell. For the SPINK2molecule, a partial molecular model containing an interaction site withKLK5 was constructed based on the sequence information and the electrondensity observed. It was confirmed that the KLK5 inhibitory peptideK51034 was bound to a region containing the KLK5 enzyme's active site(FIG. 4).

Example 5. Preparation of KLK5 Inhibitory Peptide Fc Fusion (5-1)Construction of KLK5 Inhibitory Peptide Fc Fusion Expression Vector

Using the nucleotide sequence of each inhibitory peptide (SEQ ID NO: 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31) as a template,inhibitory peptide fragments were amplified by PCR ((at 94° C. for 15seconds, at 60° C. for 30 seconds, and at 68° C. for 20 seconds)×30cycles) using the following primers and KOD-plus-(TOYOBO).

Primer 16: 5′-AGATGGGTGTTGTCTGATGACGACGGCCCTCAGTTCGGCCTGTTC-3′ (SEQ ID NO: 81, FIG. 89)  Primer 17:5′-GCAGGGGCCATTCCGGAT-3′ (SEQ ID NO: 82, FIG. 90)

Fragment B was amplified by PCR ((at 94° C. for 15 seconds, at 60° C.for 30 seconds, and at 68° C. for 10 seconds)×30 cycles) using thefollowing primers and KOD-plus-(TOYOBO).

Primer 18: 5′-AAAATCTAGAGCCGCCACCATGAAGCACCTGTGGTTCTTTCTGCTGCT-3′ (SEQ ID NO: 83, FIG. 91)  Primer 19:5′-AGACAACACCCATCTAGGAGCGGCCACCAGCAGCAGAAAGAACC-3′(SEQ ID NO: 84, FIG. 92) 

Using an Fc region of human IgG1 (SEQ ID NO: 87) as a template, fragmentC containing an Fc region of human IgG1 was amplified by PCR ((at 94° C.for 15 seconds, at 60° C. for 30 seconds, at 68° C. for 30 seconds)×30cycle) using the following primers and KOD-plus-(TOYOBO).

Primer 20: 5′-ATCCGGAATGGCCCCTGCGAACCCAAGAGCTGCGAC-3′(SEQ ID NO: 85, FIG. 93)  Primer 21:5′-AAAAGTTTAAACTCATTTGCCGGGGCTCAG-3′ (SEQ ID NO: 86, FIG. 94) 

Desired DNA fragments were amplified by overlapping PCR using theinhibitory peptide fragments amplified above, fragment B, fragment C,primer 18, primer 21, and KOD-plus-(TOYOBO).

Further, a mammalian cell expression vector, pCMA_KLK5 inhibitorypeptide Fc fusion, was constructed by cloning using restriction enzymesXbaI (NEB) and PmeI (NEB). The operation was performed according to themethod described in (1-1).

(5-2) Preparation of KLK5 Inhibitory Peptide Fc Fusion

The expression vector constructed in (5-1) was transfected into Expi293Fcells (Thermo Fisher Scientific) using PEI MAX 40000 (Polysciences), andthe culture supernatant was collected after 6 days of culturing thecells. The desired Fc fusion was collected from the culture supernatantusing MabSelect SuRe (GE healthcare), and the buffer was replaced withPBS using Amicon Ultra NMWL 10,000 (Merck KGaA Millipore), to prepare aKLK5 inhibitory peptide Fc fusion. From each clone having aglycosylation sequence in the KLK5 inhibitory peptide, the glycosylationsequence was removed by substitution of one residue, and “dN” was addedthereto as an ID showing a deglycosylated form. The modification of theglycosylation sequence does not affect the activity such as KLK5inhibitory activity or cross-reactivity at all.

(5-3) Construction of KLK5 Inhibitory Peptide Fc Fusion D1-K50055-FcExpression Vector

Using the nucleotide sequence of the KLK5 inhibitory peptide K50055 (SEQID NO: 7) as a template, inhibitory peptide fragments were amplified byPCR ((at 94° C. for 15 seconds, at 60° C. for 30 seconds, and at 68° C.for 20 seconds)×30 cycles) using the following primers andKOD-plus-(TOYOBO).

Primer 22: 5′-AGATGGGTGTTGTCTGACGGCCCTCAGTTCGGCCTGTTC-3′(SEQ ID NO: 94, FIG. 104)  Primer 17:5′-GCAGGGGCCATTCCGGAT-3′ (SEQ ID NO: 82, FIG. 90) 

Desired DNA fragments were amplified by overlapping PCR using theinhibitory peptide fragments amplified above and fragment B, fragment C,primer 18, and primer 21, which were amplified in (5-1), andKOD-plus-(TOYOBO).

Further, a mammalian cell expression vector, pCMA_KLK5 inhibitorypeptide Fc fusion, was constructed by cloning using restriction enzymesXbaI (NEB) and PmeI (NEB). The operation was performed according to themethod described in (1-1).

(5-4) Preparation of KLK5 Inhibitory Peptide Fc Fusion D1-K50055-Fc

The expression vector constructed in (5-3) was transfected into Expi293Fcells (Thermo Fisher Scientific) using PEI MAX 40000 (Polysciences), andthe culture supernatant was collected after 6 days of culturing thecells. The desired Fc fusion was collected from the culture supernatantusing MabSelect SuRe (GE healthcare), and the buffer was replaced withPBS using Amicon Ultra NMWL 10,000 (Merck KGaA Millipore), to preparethe KLK5 inhibitory peptide Fc fusion D1-K50055-Fc.

Example 6. Evaluation of KLK5 Inhibitory Peptide Fc Fusion (6-1)Evaluation of Human/Mouse KLK5, Human/Mouse KLK7, and Human/Mouse KLK14Inhibitory Activities of KLK5 Inhibitory Peptide Fc Fusion

According to the method described in Example 3-1, the inhibitoryactivities of each KLK5 inhibitory peptide Fc fusion against human/mouseKLK5, human/mouse KLK7, and human/mouse KLK14 were evaluated. As aresult of calculating the degradation rate of a substrate peptide byeach inhibitory peptide Fc fusion at each concentration and calculatingthe 50% inhibitory concentration (IC₅₀) using GraphPad Prism (version5.0; GraphPad Software Inc.) with the degradation rate of the inhibitorypeptide Fc fusion at a concentration of 0 nM taken as 100%, it wasrevealed that all of the inhibitory peptide Fc fusions inhibited humanKLK5 enzymatic activity at low concentrations (Table 4, FIG. 5, and FIG.107). Some of the inhibitory peptide Fc fusions inhibited human KLK7 orhuman KLK14 enzymatic activity at low concentrations, and some of theinhibitory peptide Fc fusions exhibited weak inhibitory activitiesagainst these proteases. The inhibitory peptide Fc fusions alsoexhibited similar activity against mouse KLK5, KLK7, or KLK14 (Table 5).The average value of three independent experiments was used to calculatethe IC₅₀ value.

TABLE 4 Human KLK5, human KLK7, or human KLK14 inhibitory activity ofeach KLK5 inhibitory peptide Fc fusion IC₅₀ (nM) for IC₅₀ (nM) for IC₅₀(nM) for ID hKLK5 hKLK7 hKLK14 D3-K51028-Fc 4.5 ± 0.1 220 ± 10  >1,000D3-K50032dN-Fc 3.9 ± 0.3 >1,000 >1,000 D3-K50055-Fc 3.7 ± 0.1 >1,000 340 ± 120 D3-K51069dN-Fc 4.5 ± 0.4 >1,000  42 ± 11 D3-K51072-Fc 4.5 ±0.3 >1,000 260 ± 60 D3-K50015-Fc 3.1 ± 0.2 >1,000  4.5 ± 1.3D3-K50016dN-Fc 4.3 ± 0.7 >1,000 >1,000 D3-K51034-Fc 5.7 ± 0.8 >1,000 960 ± 320 D3-K50062-Fc 4.3 ± 0.1 >1,000 190 ± 50 D3-K51090-Fc 4.6 ±0.5 >1,000 350 ± 30 D3-K50098dN-Fc 4.3 ± 0.3 >1,000 210 ± 60D3-K51005-Fc 8.6 ± 1.4 28 ± 3  >1,000 D3-K50031-Fc 9.5 ± 0.4 9.3 ±1.3 >1,000 D3-K51057-Fc 12 ± 1  18 ± 1  >1,000 D1-K50055-Fc 5.8 ±0.6 >1,000 >1,000

TABLE 5 Mouse KLK5, mouse KLK7, or mouse KLK14 inhibitory activity ofeach KLK5 inhibitory peptide Fc fusion IC₅₀ (nM) for IC₅₀ (nM) for IC₅₀(nM) for ID mKLK5 mKLK7 mKLK14 D3-K51028-Fc 16 110 >1,000 D3-K50032dN-Fc26 >1,000 >1,000 D3-K50055-Fc 6.8 >1,000 >1,000 D3-K51069dN-Fc 6.8 41024 D3-K51072-Fc 50 >1,000 >1,000 D3-K50015-Fc 4.4 340 48 D3-K50016dN-Fc35 >1,000 >1,000 D3-K51034-Fc 39 >1,000 >1,000 D3-K50062-Fc5.4 >1,000 >1,000 D3-K51090-Fc 5.1 730 510 D3-K50098dN-Fc 6.8 >1,000 350D3 -K51005- Fc 330 37 240 D3-K50031-Fc 270 6.3 300 D3-K51057-Fc 600 24380 D1-K50055-Fc 6.3 >1,000 >1,000

(6-2) Evaluation of Cross-Reactivity of KLK5 Inhibitory Peptide FcFusion

Like the results in (3-2), some of the inhibitory peptide Fc fusionsexhibited weak cross-reactivity to bovine trypsin, chymotrypsin, andplasmin with a final concentration of the inhibitory peptide of 1 μM(where the IC₅₀ value was less than 1 μM), but most of the inhibitorypeptides exhibited no inhibitory activity to any protease other thanKLKs (FIG. 6). Some of the inhibitory peptide Fc fusions exhibitedinhibitory activity to KLK4 or KLK12 with a final concentration of 1 μM(where the IC₅₀ value was less than 1 μM), but many of the inhibitorypeptide Fc fusions exhibited no protease inhibitory activity againstKLKs except KLK7 or KLK14. Accordingly, it was found that the inhibitorypeptide Fc fusions had high specificity like the inhibitory peptides.

(6-3) Evaluation of KLK5 Inhibitory Activity of KLK5 Inhibitory PeptideFc Fusion Using Peptide Substrate (Calculation of Inhibition ConstantK_(i))

The inhibitory activity of the KLK5 inhibitory peptide Fc fusionsagainst human KLK5 was evaluated, and the inhibition constant K_(i) wascalculated. A substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems, Inc.,ES011) was dissolved in DMSO to 10 mM and diluted with an assay buffer(50 mM Tris-HCl, 150 mM NaCl, pH 8.0) for use at a final concentrationof 25 to 200 μM. 25 μL each of human KLK5 and the KLK5 inhibitorypeptide Fc fusion diluted with the assay buffer were mixed, followed bya reaction at 37° C. for 20 minutes. Thereafter, 50 μL of a substratediluted with the assay buffer was added thereto, and the fluorescencesignal (excitation at 380 nm/emission at 460 nm) was measured usingEnspire. The human KLK5 had a final concentration of 10 nM, the KLK5inhibitory peptide Fc fusion had a final concentration of 0.5 to 25 nM,and a PROTEOSAVE (R) SS96F black plate (Sumitomo Bakelite Co., Ltd.) wasused for the reaction and the measurement.

The degradation rate of the substrate peptide by each inhibitory peptideFc fusion at each concentration was calculated, and the human KLK5inhibitory activity of each inhibitory peptide Fc fusion was evaluated,with the degradation rate of the inhibitory peptide Fc fusion at aconcentration of 0 nM taken as 100% (FIG. 102). Using GraphPad Prism(version 5.0; GraphPad Software Inc.), the maximum reaction rate V. andthe Michaelis constant K_(m) were calculated according to theMichaelis-Menten equation at an enzyme concentration of 10 nM. Further,as a result of calculating the inhibition constant K_(i) at a substrateconcentration of 100 μM using GraphPad Prism according to the Morrisonequation, it was revealed that all of the KLK5 inhibitory peptide Fcfusions inhibited human KLK5 enzymatic activity at low concentrations(Table 6). The average value of three independent experiments was usedto calculate the K_(i) value.

TABLE 6 Human KLK5 inhibition constant K_(i) of each KLK5 inhibitorypeptide Fc fusion ID K_(i) (pM) D3-K50032dN-Fc 440 ± 10 D3-K50055-Fc 190± 60 D3-K51072-Fc 360 ± 70 D3-K50016dN-Fc 360 ± 40

(6-4) Evaluation of KLK5 Inhibitory Activity of KLK5 Inhibitory PeptideFc Fusion Using Protein Substrate

Using human Desmogleinl and human Desmocollinl as protein substrates,the KLK5 inhibitory activity of each KLK5 inhibitory peptide Fc fusionwas evaluated. Human KLK5 and each KLK5 inhibitory peptide Fc fusion(D3-K50032dN-Fc, D3-K50055-Fc, D3-K51072-Fc, or D3-K50016dN-Fc) dilutedwith an assay buffer were mixed, followed by a reaction at 37° C. for 1hour. Next, each protein substrate diluted with an assay buffer wasadded thereto, followed by a reaction at 37° C. for 4 hours. Thereafter,an SDS sample buffer containing a reductant was added thereto, and theenzymatic reaction was stopped by treatment at 99° C. for 5 minutes.Thereafter, the degradation of the protein substrate was evaluated usingSDS-PAGE (reduction conditions) and Western blot analysis. Thecombinations of the substrate and enzyme, the inhibitory peptide Fcfusion, and the antibody for Western blot analysis were as follows.

Evaluation using Human Desmogleinl: hKLK5 with a final concentration of1 μM, an inhibitory peptide Fc fusion with a final concentration of0.001 to 10 μM, Recombinant Human Desmoglein-1 Fc Chimera Protein with afinal concentration of 1 μM (R&D Systems, Inc.), Desmoglein 1 Antibody(aa471-499) (LSBio), and Anti-Rabbit IgG, HRP-Linked F (ab′) 2 FragmentDonkey (GE healthcare)Evaluation using Human Desmocollinl: hKLK5 with a final concentration of0.2 μM, an inhibitory peptide Fc fusion with a final concentration of0.0002 to 2 μM, Recombinant Human Desmocollin-1 Protein with aC-terminal His tag with a final concentration of 2 μM (R&D Systems,Inc.), and Penta His HRP Conjugate (QIAGEN)

Human Desmogleinl and human Desmocollinl were not degraded in theabsence of human KLK5 but were completely degraded in the presence ofhuman KLK5. As a result of evaluating human KLK5 preincubated with eachKLK5 inhibitory peptide Fc fusion, it was revealed that each of theinhibitory peptide Fc fusions inhibited the human Desmogleinl and humanDesmocollinl degradation activities of the human KLK5 enzyme. Underconditions where the human KLK5 concentration and the inhibitory peptideFc fusion concentration were the same, the degradation of humanDesmogleinl and human Desmocollinl was completely inhibited (FIG. 103).

Example 7. Effect of KLK5 Inhibitory Peptide Fc Fusion on Suppression ofTransepidermal Water Loss (TEWL) Increase in Netherton Syndrome ModelMice (7-1) Netherton Syndrome Model Mice

Crusty2 mice having a mutation in SPINK5 that is the causative gene ofNetherton syndrome are known as Netherton syndrome model mice, andCrusty2 homomice (+/+) exhibit skin symptoms (Mutagenetix database).

(7-2) Effect of KLK5 Inhibitory Peptide Fc Fusion on Suppression of TEWLIncrease in Netherton Syndrome Model Mice

Crusty2 (+/−) mice and Crusty2 (+/+) mice were crossed by artificialinsemination, and the KLK5 inhibitory peptide Fc fusion D1-K50055-Fcproduced in (5-4) was evaluated using the resulting offspring (Crusty2(+/−) mice or Crusty2 (+/+) mice). From 0 or 1 day after birth, PBS or100 mg/kg of D1-K50055-Fc was subcutaneously administered every otherday for 4 weeks. At the first administration only, a 3-fold amount ofD1-K50055-Fc was administered as a loading dose. Using VAPO SCAN(AS-VT100RS, Asahi Techno Lab. Ltd.), TEWL on the skin of the back orhip of the mice was measured at 2 and 4 weeks after administration (FIG.7). The number of Crusty2 (+/−) mice was 9, and the number of Crusty2(+/+) mice was 12, in each of the PBS-administered group and theD1-K50055-Fc-administered group.

A statistically significant increase in TEWL was observed in Crusty2(+/+) mice as compared with Crusty2 (+/−) mice and was more remarkablein 4 week-old mice as compared with 2 week-old mice. After 4 weeks fromthe administration, a statistically significant decrease in TEWL wasobserved in both the back and the more severely affected hips in theD1-K50055-Fc-administered group as compared with the PBS-administeredgroup. From the above, it was revealed that an increase in TEWL causedby the mutation in SPINK5 was observed in the mice, and D1-K50055-Fcexhibited an inhibitory action. Further, it was shown that the peptidesof the present invention and their conjugates, including D1-K50055-Fc,are useful for reducing skin symptoms of Netherton syndrome.

INDUSTRIAL APPLICABILITY

The peptide and the conjugate provided by the present invention, and apharmaceutical composition containing the same are useful for treatingvarious diseases.

Sequence Listing Free Text

SEQ ID NO: 1: Amino acid sequence of human SPINK2 (FIG. 9)SEQ ID NO: 2: Amino acid sequence of human KLK5 (FIG. 10)SEQ ID NO: 3: Amino acid sequence of human KLK7 (FIG. 11)SEQ ID NO: 4: Amino acid sequence of human KLK14 (FIG. 12)SEQ ID NO: 5: Nucleotide sequence of the KLK5 inhibitory peptide K50032(FIG. 13)SEQ ID NO: 6: Amino acid sequence of the KLK5 inhibitory peptide K50032(FIG. 14)SEQ ID NO: 7: Nucleotide sequence of the KLK5 inhibitory peptide K50055(FIG. 15)SEQ ID NO: 8: Amino acid sequence of the KLK5 inhibitory peptide K50055(FIG. 16)SEQ ID NO: 9: Nucleotide sequence of the KLK5 inhibitory peptide K51072(FIG. 17)SEQ ID NO: 10: Amino acid sequence of the KLK5 inhibitory peptide K51072(FIG. 18)SEQ ID NO: 11: Nucleotide sequence of the KLK5 inhibitory peptide K50016(FIG. 19)SEQ ID NO: 12: Amino acid sequence of the KLK5 inhibitory peptide K50016(FIG. 20)SEQ ID NO: 13: Nucleotide sequence of the KLK5 inhibitory peptide K51034(FIG. 21)SEQ ID NO: 14: Amino acid sequence of the KLK5 inhibitory peptide K51034(FIG. 22)SEQ ID NO: 15: Nucleotide sequence of the KLK5 inhibitory peptide K50062(FIG. 23)SEQ ID NO: 16: Amino acid sequence of the KLK5 inhibitory peptide K50062(FIG. 24)SEQ ID NO: 17: Nucleotide sequence of the KLK5 inhibitory peptide K51090(FIG. 25)SEQ ID NO: 18: Amino acid sequence of the KLK5 inhibitory peptide K51090(FIG. 26)SEQ ID NO: 19: Nucleotide sequence of the KLK5 inhibitory peptide K50098(FIG. 27)SEQ ID NO: 20: Amino acid sequence of the KLK5 inhibitory peptide K50098(FIG. 28)SEQ ID NO: 21: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideK51028 (FIG. 29)SEQ ID NO: 22: Amino acid sequence of the KLK5/KLK7 inhibitory peptideK51028 (FIG. 30)SEQ ID NO: 23: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideK51005 (FIG. 31)SEQ ID NO: 24: Amino acid sequence of the KLK5/KLK7 inhibitory peptideK51005 (FIG. 32)SEQ ID NO: 25: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideK50031 (FIG. 33)SEQ ID NO: 26: Amino acid sequence of the KLK5/KLK7 inhibitory peptideK50031 (FIG. 34)SEQ ID NO: 27: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideK51057 (FIG. 35)SEQ ID NO: 28: Amino acid sequence of the KLK5/KLK7 inhibitory peptideK51057 (FIG. 36)SEQ ID NO: 29: Nucleotide sequence of the KLK5/KLK14 inhibitory peptideK51069 (FIG. 37)SEQ ID NO: 30: Amino acid sequence of the KLK5/KLK14 inhibitory peptideK51069 (FIG. 38)SEQ ID NO: 31: Nucleotide sequence of the KLK5/KLK14 inhibitory peptideK50015 (FIG. 39)SEQ ID NO: 32: Amino acid sequence of the KLK5/KLK14 inhibitory peptideK50015 (FIG. 40)SEQ ID NO: 33: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50032dN-Fc (FIG. 41)SEQ ID NO: 34: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50032dN-Fc (FIG. 42)SEQ ID NO: 35: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50055-Fc (FIG. 43)SEQ ID NO: 36: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50055-Fc (FIG. 44)SEQ ID NO: 37: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K51072-Fc (FIG. 45)SEQ ID NO: 38: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K51072-Fc (FIG. 46)SEQ ID NO: 39: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50016dN-Fc (FIG. 47)SEQ ID NO: 40: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50016dN-Fc (FIG. 48)SEQ ID NO: 41: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K51034-Fc (FIG. 49)SEQ ID NO: 42: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K51034-Fc (FIG. 50)SEQ ID NO: 43: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50062-Fc (FIG. 51)SEQ ID NO: 44: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50062-Fc (FIG. 52)SEQ ID NO: 45: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K51090-Fc (FIG. 53)SEQ ID NO: 46: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K51090-Fc (FIG. 54)SEQ ID NO: 47: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D3-K50098dN-Fc (FIG. 55)SEQ ID NO: 48: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D3-K50098dN-Fc (FIG. 56)SEQ ID NO: 49: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51028-Fc (FIG. 57)SEQ ID NO: 50: Amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51028-Fc (FIG. 58)SEQ ID NO: 51: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51005-Fc (FIG. 59)SEQ ID NO: 52: Amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51005-Fc (FIG. 60)SEQ ID NO: 53: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K50031-Fc (FIG. 61)SEQ ID NO: 54: Amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K50031-Fc (FIG. 62)SEQ ID NO: 55: Nucleotide sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51057-Fc (FIG. 63)SEQ ID NO: 56: Amino acid sequence of the KLK5/KLK7 inhibitory peptideFc fusion D3-K51057-Fc (FIG. 64)SEQ ID NO: 57: Nucleotide sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K51069dN-Fc (FIG. 65)SEQ ID NO: 58: Amino acid sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K51069dN-Fc (FIG. 66)SEQ ID NO: 59: Nucleotide sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K50015-Fc (FIG. 67)SEQ ID NO: 60: Amino acid sequence of the KLK5/KLK14 inhibitory peptideFc fusion D3-K50015-Fc (FIG. 68)SEQ ID NO: 61: Formula of the SPINK2 mutant peptide (FIG. 69)SEQ ID NO: 62: Nucleotide sequence of primer 1 (FIG. 70)SEQ ID NO: 63: Nucleotide sequence of primer 2 (FIG. 71)SEQ ID NO: 64: Nucleotide sequence of primer 3 (FIG. 72)SEQ ID NO: 65: Nucleotide sequence of primer 4 (FIG. 73)SEQ ID NO: 66: Nucleotide sequence of primer 5 (FIG. 74)SEQ ID NO: 67: Nucleotide sequence of primer 6 (FIG. 75)SEQ ID NO: 68: Nucleotide sequence of primer 7 (FIG. 76)SEQ ID NO: 69: Nucleotide sequence of primer 8 (FIG. 77)SEQ ID NO: 70: Nucleotide sequence of primer 9 (FIG. 78)SEQ ID NO: 71: Nucleotide sequence of primer 10 (FIG. 79)SEQ ID NO: 72: Nucleotide sequence of primer 11 (FIG. 80)SEQ ID NO: 73: Nucleotide sequence of primer 12 (FIG. 81)SEQ ID NO: 74: Nucleotide sequence of primer 13 (FIG. 82)SEQ ID NO: 75: Nucleotide sequence of primer 14 (FIG. 83)SEQ ID NO: 76: Nucleotide sequence of primer 15 (FIG. 84)SEQ ID NO: 77: Amino acid sequence in the KLK7 substrate peptide (FIG.85)SEQ ID NO: 78: Amino acid sequence in the bovine c-chymotrypsinsubstrate peptide (FIG. 86)SEQ ID NO: 79: Amino acid sequence in the neutrophil elastase substratepeptide (FIG. 87)SEQ ID NO: 80: Amino acid sequence in the human protein C substratepeptide (FIG. 88)SEQ ID NO: 81: Nucleotide sequence of primer 16 (FIG. 89)SEQ ID NO: 82: Nucleotide sequence of primer 17 (FIG. 90)SEQ ID NO: 83: Nucleotide sequence of primer 18 (FIG. 91)SEQ ID NO: 84: Nucleotide sequence of primer 19 (FIG. 92)SEQ ID NO: 85: Nucleotide sequence of primer 20 (FIG. 93)SEQ ID NO: 86: Nucleotide sequence of primer 21 (FIG. 94)SEQ ID NO: 87: Amino acid sequence of Fc of human IgG1 (FIG. 95)SEQ ID NO: 88: Amino acid sequence of D8 of human SPINK5 (FIG. 96)SEQ ID NO: 89: Amino acid sequence of D9 of human SPINK5 (FIG. 97)SEQ ID NO: 90: Amino acid sequence of human SPINK9 (FIG. 98)SEQ ID NO: 91: Amino acid sequence of mouse KLK5 (FIG. 99)SEQ ID NO: 92: Amino acid sequence of mouse KLK7 (FIG. 100)SEQ ID NO: 93: Amino acid sequence of mouse KLK14 (FIG. 101)SEQ ID NO: 94: Nucleotide sequence of primer 22 (FIG. 104)SEQ ID NO: 95: Nucleotide sequence of the KLK5 inhibitory peptide Fcfusion D1-K50055-Fc (FIG. 105)SEQ ID NO: 96: Amino acid sequence of the KLK5 inhibitory peptide Fcfusion D1-K50055-Fc (FIG. 106)

1. A SPINK2 mutant peptide that inhibits the protease activity of activehuman KLK5, wherein the peptide comprises: (i) the amino acid sequenceset forth in any one of SEQ ID Nos: 6, 8, 10, 12, 14, 16, 18, and 20; or(ii) the amino acid sequence consisting of amino acids 4 to 66 of theamino acid sequence set forth in any one of SEQ ID Nos: 34, 40 and 48.2. A conjugate comprising the peptide according to claim 1, wherein thepeptide has at least one moiety attached to the peptide.
 3. Theconjugate according to claim 2, wherein the at least one moietycomprises a second peptide that is not the SPINK2 mutant.
 4. Theconjugate according to claim 3, wherein the second peptide is located onthe amino terminal side of the SPINK2 mutant.
 5. The conjugate accordingto claim 3, wherein the second peptide is located on the carboxylterminal side of the SPINK2 mutant.
 6. The conjugate according to claim5, wherein the second peptide is an antibody or a fragment thereof andcomprises one or more Fc regions.
 7. The conjugate according to claim 6,wherein each Fc region is an Fc region of human immunoglobulin, or afragment thereof.
 8. The conjugate according to claim 6, wherein each Fcregion is an Fc region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD,or IgE, or a fragment thereof.
 9. The conjugate according to claim 6,wherein each Fc region is an Fc region of human IgG1, or a fragmentthereof.
 10. The conjugate according to claim 9, wherein each Fc regionof human IgG1 comprises the amino acid sequence set forth in SEQ ID NO:87.
 11. The conjugate according to claim 6, wherein each Fc region is awild type or a mutant Fc region.
 12. The conjugate according to claim 2,wherein the conjugate comprises one or more aspartic acids and/orglutamic acids added to the amino terminus thereof.
 13. The conjugateaccording to claim 2, wherein the SPINK2 mutant and the second peptideare linked to each other via a linker.
 14. The conjugate according toclaim 13, wherein the linker is a third peptide that is not the SPINK2mutant or the second peptide.
 15. A composition comprising the peptideaccording to claim
 1. 16. A composition comprising the conjugateaccording to claim
 2. 17. A pharmaceutical composition comprising thepeptide according to claim
 1. 18. The pharmaceutical compositionaccording to claim 17, further comprising an additional pharmaceuticalproduct.
 19. A pharmaceutical composition comprising the conjugateaccording to claim
 2. 20. The pharmaceutical composition according toclaim 19, further comprising an additional pharmaceutical product. 21.The conjugate according to claim 2, wherein the conjugate comprises: (i)the amino acid sequence set forth in any one of SEQ ID Nos: 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 96; or (ii) the aminoacid sequence formed by the deletion of one amino acid from the aminoacid sequence according to (i); wherein the conjugate inhibits theprotease activity of KLK5.
 22. A conjugate comprising the amino acidsequence set forth in SEQ ID No: 34 or the amino acid sequence formed bythe deletion of one amino acid from the amino acid sequence set forth inSEQ ID No:
 34. 23. A composition comprising the conjugate according toclaim
 22. 24. The composition according to claim 23, wherein thecomposition is a pharmaceutical composition.
 25. A conjugate comprisingthe amino acid sequence set forth in SEQ ID No: 36 or the amino acidsequence formed by the deletion of one amino acid from the amino acidsequence set forth in SEQ ID No:
 36. 26. A composition comprising theconjugate according to claim
 25. 27. The composition according to claim26, wherein the composition is a pharmaceutical composition.
 28. Aconjugate comprising the amino acid sequence set forth in SEQ ID No: 38or the amino acid sequence formed by the deletion of one amino acid fromthe amino acid sequence set forth in SEQ ID No:
 38. 29. A compositioncomprising the conjugate according to claim
 28. 30. The compositionaccording to claim 29, wherein the composition is a pharmaceuticalcomposition.